Induced Hepatocytes and Uses Thereof

ABSTRACT

Disclosed herein are induced hepatocytes from a trophoblast stem cell, methods for inducing the cells, and compositions thereof. Also disclosed herein are methods of treating a disease or disorder (e.g., liver-associated) by utilizing an induced hepatocyte disclosed herein.

CROSS-REFERENCE

This application is a divisional application of U.S. application Ser.No. 14/952,023, filed Nov. 25, 2015, which claims the benefit of U.S.Provisional Application No. 62/085,185, filed on Nov. 26, 2014, each ofwhich is incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 31, 2015, isnamed 44980-704.201_SL.txt and is 31,606 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications disclosed herein areincorporated by reference to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference. In the event ofa conflict between a term disclosed herein and a term in an incorporatedreference, the term herein controls.

BRIEF SUMMARY

In one of many aspects, disclosed herein is a method of inducing atrophoblast stem (TS) cell to differentiate into an induced hepatocytein vitro, comprising: contacting the trophoblast stem cell with aconditioned medium (e.g., for sufficient time) to induce differentiationof the trophoblast stem cell into an induced hepatocyte, wherein thecondition medium comprises a fibroblast growth factor (FGF), a steroid,and a cytokine. In some embodiments, disclosed herein is a method ofinducing a trophoblast stem (TS) cell to differentiate into an inducedhepatocyte in vitro, which comprises (a) contacting the trophoblast stemcell in a conditioned medium comprising a fibroblast growth factor(FGF), a steroid, and a cytokine; and (b) incubating the cell forsufficient time to induce differentiation of the trophoblast stem cellinto an induced hepatocyte. In some embodiments, the method furthercomprises contacting the trophoblast stem cell with the FGF prior toaddition of the steroid and the cytokine to the conditioned medium. Insome embodiments, the trophoblast stem cell is contacted with FGF for atleast 2 hours, at least 4 hours, at least 6 hours, 8 hours, at least 12hours, at least 16 hours, at least 20 hours, or at least 24 hours priorto addition of the steroid and the cytokine to the conditioned medium.In some embodiments, the method further comprises incubating thetrophoblast stem cell for at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, or at least 7days. In some embodiments, the steroid and the cytokine are addedsimultaneously or sequentially into the conditioned medium.

In some embodiments, an induced hepatocyte herein is a hepaticprogenitor cell. In some embodiments, FGF upregulates miRNA-124a in theTS cell. In some embodiments, elevated level of miRNA-124a initiatesdefinitive endoderm (DE) specification in the TS cell. In someembodiments, the DE specification is associated with biomarkerscomprising forkhead box protein A2 (FOXA2), SRY-box 17 (SOX17),Goosecoid (GSC), or Homeodomain protein MIXL1. In some embodiments, theDE specification is associated with elevated expression levels of SOX17,FOXA2, and GSC. In some embodiments, the elevated expression levels areincreased protein expression levels. In some embodiments, the DEspecification is associated with a decreased expression level of MIXL1.In some embodiments, the decreased expression level is a decreasedprotein expression levels. In some embodiments, the elevated proteinexpression levels of SOX17, FOXA2, and GSC and the decreased proteinexpression level of MIXL1 are relative to the protein expression levelsof SOX17, FOXA2, GSC, and MIXL1 in an equivalent TS cell that has notundergone DE specification. In some embodiments, the DE specification isfurther associated with elevated expression levels of SOX2, NANOG, andOCT4. In some embodiments, elevated expression levels of SOX2, NANOG,and OCT4 are increased level of protein expressions. In someembodiments, elevated expression levels of SOX2, NANOG, and OCT4 areincreased level of gene expressions. In some embodiments, the elevatedexpression levels of SOX2, NANOG, and OCT4 are relative to theexpression levels of SOX2, NANOG, and OCT4 in an equivalent TS cell thathas not undergone DE specification. In some embodiments, differentiationinduced by a method herein comprises one or more of four stages:primitive streak to definitive endoderm (DE) stage, hepatic specifiedendoderm stage, hepatoblastic stage, and the fetal and adult hepatocytecell stage. In some embodiments, one or more biomarkers selected fromthe group consisting of CXCR4, FOXA2, SOX17, HHEX, TTR, ALB, TAT,CYP7A1, BSEP, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, HNF4α, and anycombination thereof express in one or more of the four stages. In someembodiments, one or more biomarkers selected from the group consistingof CXCR4, FOXA2, SOX17, HHEX, and any combination thereof, express atthe primitive streak to DE stage. In some embodiments, an expressionlevel of CXCR4, FOXA2, SOX17, and/or HHEX increases at the primitivestreak to DE stage, relative to that before the primitive streak to DEstage. In some embodiments, the increased expression level is anincreased level of gene expression. In some embodiments, the expressionlevel of CXCR4, FOXA2, SOX17 and/or HHEX increases by about 1 fold andabout 10,000 fold higher than that before the primitive streak to DEstage. In some embodiments, the expression level of CXCR4, FOXA2, SOX17and/or HHEX increases by about 10 fold and about 1000 fold higher thanthat before the primitive streak to DE stage. In some embodiments, oneor more biomarkers selected from the group consisting of SOX17, TTR,ALB, TAT, SERPINA1, CYP7A1, and any combination thereof express in thehepatic specified endoderm stage. In some embodiments, an expressionlevel of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1 increases at thehepatic specified endoderm stage, relative to that before the hepaticspecified endoderm stage. In some embodiments, the increased expressionlevel is an increased level of gene expression. In some embodiments, theexpression level of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1increases by about 1 fold and about 1000 fold higher than that beforethe hepatic specified endoderm stage. In some embodiments, theexpression level of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1increases by about 10 fold and about 100 fold higher than that beforethe hepatic specified endoderm stage. In some embodiments, one or morebiomarkers selected from the group consisting of TTR, ALB, TAT, CYP7A1,SERPINA1, BSEP, and any combination thereof express at the hepatoblasticstage. In some embodiments, an expression level of TTR, ALB, TAT,CYP7A1, SERPINA1, and/or BSEP increases at the hepatoblastic stage,relative to that before the hepatoblastic stage. In some embodiments,the increased expression level is an increased level of gene expression.In some embodiments, the expression level of TTR, ALB, TAT, CYP7A1,SERPINA1, and/or BSEP increases by about 1 fold and about 1000 foldhigher than that before the hepatoblastic stage. In some embodiments,the expression level of TTR, ALB, TAT, CYP7A1, SERPINA1, and/or BSEPincreases by about 10 fold and about 100 fold higher than that beforethe hepatoblastic stage. In some embodiments, one or more biomarkersselected from the group consisting of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, HNF4α, and any combination thereofexpress at the fetal and adult hepatocyte-like cell stage. In someembodiments, an expression level of HHEX, BSEP, TTR, ALB, TAT, SERPINA1,G6PC, ABCC2, C/EBPβ, HNF1α, and/or HNF4α increases at the fetal andadult hepatocyte-like cell stage, relative to that before the fetal andadult hepatocyte-like cell stage. In some embodiments, the increasedexpression level is an increased level of gene expression. In someembodiments, the expression level of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and/or HNF4α increases by about 10fold and about 1000 fold higher than that before the fetal and adulthepatocyte cell stage. In some embodiments, the expression level ofHHEX, BSEP, TTR, ALB, TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and/orHNF4α increases by at least 100 fold higher than that before the fetaland adult hepatocyte cell stage.

In some embodiments, a trophoblast stem cell herein is a humantrophoblast stem cell. In some embodiments, the steroid isdexamethasone. In some embodiments, the cytokine is oncostatin M. Insome embodiments, the oncostatin M is a human oncostatin M. In someembodiments, the human oncostatin M is a recombinant human oncostatin M.In some embodiments, the conditioned medium further comprises a bonemorphogenetic protein (BMP). In some embodiments, the BMP is present ina concentration of about 1-100 ng/ml. In some embodiments, the BMP ispresent in a concentration of about 1-50 ng/ml, e.g., about 20 ng/ml. Insome embodiments, the BMP is BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,BMP8a, BMP8b, BMP10, or BMP15. In some embodiments, the BMP is BMP4. Insome embodiments, the conditioned medium further comprises a hepaticgrowth factor (HGF). In some embodiments, the HGF is present in aconcentration of about: 0.1-50 ng/ml or 0.1-25 ng/ml, e.g., about 5ng/ml.

In some embodiments, a hepatocyte disclosed herein is immune privileged.In some embodiments, the induced hepatocyte expresses TGFβ1. In someembodiments, the induced hepatocyte expresses TGFβ1, fibronectin, andcollagen IV in extracellular matrix (ECM). In some embodiments, theinduced hepatocyte expresses HLA-G. In some embodiments, the inducedhepatocyte expresses HLA-G and stem-121. In some embodiments, theinduced hepatocytes recruit CD4⁺Foxp3⁺ Treg cells. In some embodiments,the induced hepatocytes form tissue of a 3-dimensional structure. Insome embodiments, the induced hepatocytes cluster or aggregate. In someembodiments, the induced hepatocytes form a crescent cell mass. In someembodiments, the induced hepatocytes comprise a peripheral compartmentand a central compartment. In some embodiments, the induced hepatocytesdistribute irregularly along ECM beyond basement membrane in theperipheral compartment. In some embodiments, the induced hepatocytesdistribute from basal towards central areas in the central compartment.In some embodiments, the induced hepatocyte expresses one or moremarkers selected from the group consisting of TGFβ1, HLA-G, stem 121,C-kit, CK19, CK18, ALB, α-AFP, betatrophin, ADH1, APOF, CPS1, GATA4,CYP1A1, CYP2B6, ASGR1, CXCR4, BSEP, MRP2, Cx32, and any combinationthereof. In some embodiments, the induced hepatocyte expresses one ormore markers selected from the group consisting of TGFβ1, HLA-G, stem121, C-kit, betatrophin, ADH1, APOF, CPS1, CYP2B6, ASGR1, CXCR4, Cx32,and any combination thereof. In some embodiments, the induced hepatocyteexpresses one or more markers selected from the group consisting ofCPS1, CYP2B6, and a combination thereof.

In one aspect, provided herein is an induced hepatocyte produced by anymethod disclosed herein. In another aspect, provided herein is anisolated induced hepatocyte derived from a trophoblast stem cell. Inanother aspect, provided herein is an isolated hepatocyte induced from atrophoblast stem cell. In some embodiments, the hepatocyte expresses oneor more biomarkers selected from the group consisting of transforminggrowth factor beta 1 (TGFβ1), human leukocyte antigen G (HLA-G), clusterof differentiation 4 (CD4), forkhead box P3 (Foxp3), human cytoplasmicmarker stem 121 (stem 121), mast/stem cell growth factor receptor C-kit(C-kit), betatrophin, apolipoprotein F (APOF), alcohol dehydrogenase-1(ADH1), carbamoyl-phosphate synthase 1 (CPS1), GATA transcription factor4 (GATA4), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1),cytochrome P450 2B6 (CYP2B6), asialoglycoprotein receptor 1 (ASGR1),C—X—C chemokine receptor type 4 (CXCR4), bile salt export pump (BSEP),multi-drug resistance protein-2 (MRP2), connexin 32 (CX32), forkhead boxprotein A2 (FOXA2), SRY-box 17 (SOX17), hexosaminidase A alphapolypeptide (HEXA), hematopoietically expressed homeobox (HHEX),transthyretin (TTR), albumin (ALB), tyrosine aminotransferase (TAT),cytochrome P450 7A1 (CYP7A1), glucose-6-phosphatase (G6PC), serpinpeptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member1 (SERPINA1), ATP-binding cassette sub-family C (ABCC2),CCAAT-enhancer-binding protein beta (C/EBPβ), hepatocyte nuclear factor1-alpha (HNF1α), hepatocyte nuclear factor 4-alpha (HNF4α),alpha-1-fetoprotein (AFP), cytokeratin 8 (CK8), phosphoenolpyruvatecarboxykinase 2 mitochondrial (PCK2), glycogen synthase 2 (GYS2),hepatocyte nuclear factor 6 (HNF6), alcohol dehydrogenase 1C (class I)gamma polypeptide (ADH1C), cytochrome P450 3A4 (CYP3A4), prosperohomeobox 1 (PROX1), tryptophan 2,3-dioxygenase (TDO2), cytokeratin 18(CK18), and cytokeratin 19 (CK19).

In some embodiments, a hepatocyte herein is a hepatic progenitor cell.In some embodiments, FGF upregulates miRNA-124a in the TS cell. In someembodiments, elevated level of miRNA-124a initiates definitive endoderm(DE) specification in the TS cell. In some embodiments, the DEspecification is associated with biomarkers comprising forkhead boxprotein A2 (FOXA2), SRY-box 17 (SOX17), Goosecoid (GSC), or Homeodomainprotein MIXL1. In some embodiments, the DE specification is associatedwith elevated expression levels of SOX17, FOXA2, and GSC. In someembodiments, the elevated expression levels are increased proteinexpression levels. In some embodiments, the DE specification isassociated with a decreased expression level of MIXL1. In someembodiments, the decreased expression level is a decreased proteinexpression levels. In some embodiments, the elevated protein expressionlevels of SOX17, FOXA2, and GSC and the decreased protein expressionlevel of MIXL1 are relative to the protein expression levels of SOX17,FOXA2, GSC, and MIXL1 in an equivalent TS cell that has not undergone DEspecification. In some embodiments, the DE specification is furtherassociated with elevated expression levels of SOX2, NANOG, and OCT4. Insome embodiments, elevated expression levels of SOX2, NANOG, and OCT4are increased level of protein expressions. In some embodiments,elevated expression levels of SOX2, NANOG, and OCT4 are increased levelof gene expressions. In some embodiments, the elevated expression levelsof SOX2, NANOG, and OCT4 are relative to the expression levels of SOX2,NANOG, and OCT4 in an equivalent TS cell that has not undergone DEspecification. In some embodiments, a hepatocyte disclosed herein is atone of four stages: primitive streak to definitive endoderm (DE) stage,hepatic specified endoderm stage, hepatoblastic stage, and the fetal andadult hepatocyte cell stage. In some embodiments, one or more biomarkersselected from the group consisting of CXCR4, FOXA2, SOX17, HHEX, TTR,ALB, TAT, CYP7A1, BSEP, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, HNF4α, andany combination thereof express in one or more of the four stages. Insome embodiments, one or more biomarkers selected from the groupconsisting of CXCR4, FOXA2, SOX17, HHEX, and any combination thereof,express at the primitive streak to DE stage. In some embodiments, anexpression level of CXCR4, FOXA2, SOX17, and/or HHEX increases at theprimitive streak to DE stage, relative to that before the primitivestreak to DE stage. In some embodiments, the increased expression levelis an increased level of gene expression. In some embodiments, theexpression level of CXCR4, FOXA2, SOX17 and/or HHEX increases by about 1fold and about 10,000 fold higher than that before the primitive streakto DE stage. In some embodiments, the expression level of CXCR4, FOXA2,SOX17 and/or HHEX increases by about 10 fold and about 1000 fold higherthan that before the primitive streak to DE stage. In some embodiments,one or more biomarkers selected from the group consisting of SOX17, TTR,ALB, TAT, SERPINA1, CYP7A1, and any combination thereof express in thehepatic specified endoderm stage. In some embodiments, an expressionlevel of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1 increases at thehepatic specified endoderm stage, relative to that before the hepaticspecified endoderm stage. In some embodiments, the increased expressionlevel is an increased level of gene expression. In some embodiments, theexpression level of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1increases by about 1 fold and about 1000 fold higher than that beforethe hepatic specified endoderm stage. In some embodiments, theexpression level of SOX17, TTR, ALB, TAT, SERPINA1, and/or CYP7A1increases by about 10 fold and about 100 fold higher than that beforethe hepatic specified endoderm stage. In some embodiments, one or morebiomarkers selected from the group consisting of TTR, ALB, TAT, CYP7A1,SERPINA1, bile salts excretion pump (BSEP), and any combination thereofexpress at the hepatoblastic stage. In some embodiments, an expressionlevel of TTR, ALB, TAT, CYP7A1, SERPINA1, and/or BSEP increases at thehepatoblastic stage, relative to that before the hepatoblastic stage. Insome embodiments, the increased expression level is an increased levelof gene expression. In some embodiments, the expression level of TTR,ALB, TAT, CYP7A1, SERPINA1, and/or BSEP increases by about 1 fold andabout 1000 fold higher than that before the hepatoblastic stage. In someembodiments, the expression level of TTR, ALB, TAT, CYP7A1, SERPINA1,and/or BSEP increases by about 10 fold and about 100 fold higher thanthat before the hepatoblastic stage. In some embodiments, one or morebiomarkers selected from the group consisting of HHEX, BSEP, TTR, ALB,TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, HNF4α, and any combinationthereof express at the fetal and adult hepatocyte-like cell stage. Insome embodiments, an expression level of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and/or HNF4α increases at thefetal and adult hepatocyte-like cell stage, relative to that before thefetal and adult hepatocyte-like cell stage. In some embodiments, theincreased expression level is an increased level of gene expression. Insome embodiments, the expression level of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and/or HNF4α increases by about 10fold and about 1000 fold higher than that before the fetal and adulthepatocyte cell stage. In some embodiments, the expression level ofHHEX, BSEP, TTR, ALB, TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and/orHNF4α increases by at least 100 fold higher than that before the fetaland adult hepatocyte cell stage. In some embodiments, the trophoblaststem cell is a human trophoblast stem cell.

In some embodiments, a hepatocyte herein is immune privileged. In someembodiments, the hepatocyte expresses TGFβ1. In some embodiments, thehepatocyte expresses TGFβ1, fibronectin, and collagen IV inextracellular matrix (ECM). In some embodiments, the hepatocyteexpresses HLA-G. In some embodiments, the hepatocyte expresses HLA-G andstem-121. In some embodiments, the hepatocyte recruits CD4⁺Foxp3⁺ Tregcells. In some embodiments, the hepatocytes form tissue of a3-dimensional structure. In some embodiments, the hepatocytes cluster oraggregate. In some embodiments, the hepatocytes form a crescent cellmass. In some embodiments, the hepatocytes comprise a peripheralcompartment and a central compartment. In some embodiments, thehepatocytes distribute irregularly along ECM beyond basement membrane inthe peripheral compartment. In some embodiments, the hepatocytesdistribute from basal towards central areas in the central compartment.In some embodiments, the hepatocyte expresses one or more markersselected from the group consisting of TGFβ1, HLA-G, stem 121, C-kit,CK19, CK18, ALB, α-AFP, betatrophin, ADH1, APOF, CPS1, GATA4, CYP1A1,CYP2B6, ASGR1, CXCR4, BSEP, MRP2, Cx32, and any combination thereof. Insome embodiments, the hepatocyte expresses one or more markers selectedfrom the group consisting of TGFβ1, HLA-G, stem 121, C-kit, betatrophin,ADH1, APOF, CPS1, CYP2B6, ASGR1, CXCR4, Cx32, and any combinationthereof. In some embodiments, the hepatocyte expresses one or moremarkers selected from the group consisting of CPS1, CYP2B6, and acombination thereof. In some embodiments, the hepatocyte expresses oneor more markers selected from the group consisting of stem 121, C-kit,CK19, CK18, and any combination thereof. In some embodiments, thehepatocyte expresses one or more markers selected from the groupconsisting of ALB, AFP, betatrophin, ADH1, APOF, CPS1, GATA4, CYP1A1,CYP2B6, and any combination thereof. In some embodiments, the hepatocyteexpresses one or more markers selected from the group consisting ofASGR1, CXCR4, BSEP, MRP2, Cx32, and any combination thereof.

Also disclosed herein is a method of screening a therapeutic compoundfor use in treatment or prevention of a condition, comprising:contacting an isolated hepatocyte disclosed herein with the therapeuticcompound; and detecting an expression level of a biomarker in theisolated hepatocyte. In some embodiments, the expression level of abiomarker in the isolated hepatocyte increases as compared to anequivalent isolated hepatocyte not contacted with the therapeuticcompound. In some embodiments, the expression level of a biomarker inthe isolated hepatocyte decreases as compared to an equivalent isolatedhepatocyte not contacted with the therapeutic compound. In someembodiments, the expression level is a gene expression level. In someembodiments, the biomarker comprises CYP1A2, CYP2B6, CYP2C8, CYP2C9,CYP2D6, CYP2E1, CYP3A4, or CYP7A1. In some embodiments, the therapeuticcompound is a small molecule drug, a peptide, or a protein. In someembodiments, the therapeutic compound is a synthetic chemical drug. Insome embodiments, the condition is a liver failure. In some embodiments,the condition is a liver-associated disease or disorder. In someembodiments, the liver-associated disease or disorder comprises alagillesyndrome, alpha 1 anti-trypsin deficiency, autoimmune hepatitis, benignliver tumors, biliary atresia, cirrhosis, cystic disease of the liver,fatty liver disease including alcohol-related liver disease andnon-alcohol fatty liver disease (NAFLD), galactosemia, gallstones,Gilbert's Syndrome, hemochromatosis, liver cysts, liver cancer, liverdisease in pregnancy (optionally acute fatty liver of pregnancy,intrahepatic cholestasis of pregnancy, preeclampsia, or HELLP Syndrome(hemolysis, elevated liver tests, low platelets)), neonatal hepatitis,primary billary cirrhosis, primary sclerosing cholangitis, porphyria,Reye's Syndrome, sarcoidosis, toxic hepatitis, type 1 glocogen storagedisease, tyrosinemia, viral hepatitis, Wilson disease, or anycombination thereof.

In one aspect, disclosed herein is a composition (e.g., pharmaceuticalcomposition) comprising any hepatocyte disclosed herein.

In another aspect, disclosed herein is a method of treating a conditionin a subject, comprising administering to a subject a pharmaceuticalcomposition that comprises an isolated hepatocyte herein, in an amounteffective for the hepatocytes to engraft to the subject (e.g., to thesubject's liver). In some embodiments, the hepatocytes are administeredin a pharmaceutically acceptable carrier. In some embodiments, thepharmaceutically acceptable carrier comprises a phosphate buffer saline.In some embodiments, the hepatocytes are administered in a suspensioncontaining about 1×10⁶ to about 100×10⁶ cells per ml, about 1×10⁶ toabout 250×10⁶ cells per ml, about 1×10⁶ to about 500×10⁶ cells per ml,or about 10×10⁶ to about 40×10⁶ cells per ml. In some embodiments, thehepatocytes are administered in a volume of about: 1-5 ml, 1-10 ml, 1-50ml, 1-100 ml, or 10-150 ml. In some embodiments, the subject is a human.In some embodiments, the administering comprises an injection, e.g.,intravenous injection. In some embodiments, the injection isadministered at a hepatic vein. In some embodiments, the injection isadministered at a hepatic artery. In some embodiments, the condition isa liver-associated disease or disorder. In some embodiments, thecondition is a liver failure. In some embodiments, the liver-associateddisease or disorder comprises alagille syndrome, alpha 1 anti-trypsindeficiency, autoimmune hepatitis, benign liver tumors, biliary atresia,cirrhosis, cystic disease of the liver, fatty liver disease includingalcohol-related liver disease and non-alcohol fatty liver disease(NAFLD), galactosemia, gallstones, Gilbert's Syndrome, hemochromatosis,liver cysts, liver cancer, liver disease in pregnancy (optionally, acutefatty liver of pregnancy, intrahepatic cholestasis of pregnancy,preeclampsia, or HELLP Syndrome (hemolysis, elevated liver tests, lowplatelets)), neonatal hepatitis, primary billary cirrhosis, primarysclerosing cholangitis, porphyria, Reye's Syndrome, sarcoidosis, toxichepatitis, type 1 glocogen storage disease, tyrosinemia, viralhepatitis, Wilson disease, or any combination thereof.

In another aspect, disclosed herein is a use of a composition comprisinga hepatocyte herein for the production of therapeutic proteins. In someembodiments, the therapeutic proteins comprise major plasma proteinssuch as human serum albumin, soluble plasma fibronectin, α-fetoprotein,C-reactive protein, and several globulins; proteins involved inhemostasis and fibrinolysis such as coagulation factors involved in thecoagulation cascade, α2-macroglobulin, α1-antitrypsin, antithrombin III,protein S, protein C, plasminogen, α2-antiplasmin, and complementcomponent 3; carrier proteins such as albumin, ceruloplasmin,transcortin, haptoglobin, hemopexin, IGF binding rotein, major urinaryproteins, retinol binding protein, sex hormone-binding globulin,transthyretin, transferrin, and Vitamin D-binding protein; hormones suchas insulin-like growth factor 1, thrombopoietin, hepcidin, andbetatrophin; prohormones such as angiotensinogen; or apolipoproteins.

Also disclosed herein is a use of a composition comprising a hepatocyteherein for liver regeneration. In some embodiments, the liverregeneration is an ex vivo liver regeneration. In some embodiments, theex vivo liver regeneration is a bioprinting method. In some embodiments,the bioprinting method is a 3 dimensional bioprinting method.

In one aspect, disclosed herein is a use of a composition comprising ahepatocyte herein for bioprinting. In some embodiments, the bioprintingis a 3 dimensional bioprinting.

Also disclosed herein is a use of the hepatocyte herein for tissuescaffold generation. In some embodiments, the tissue scaffold is a 3dimensional tissue scaffold.

In one aspect, disclosed herein is a use of a composition comprising ahepatocyte herein for gene therapy. In some embodiments, the genetherapy is an ex vivo gene therapy.

In another aspect, disclosed herein is an artificial tissue generatedfrom hepatocytes herein. In some embodiments, the tissue isthree-dimensional. In some embodiments, the issue is vascularized.

Also disclosed herein is an artificial organ generated from hepatocytesherein.

In some embodiments, a hepatocyte, tissue, or organ disclosed hereinproduces AFP, ALB, alpha-1-antitrypsin, glucose, or glycogen. In someembodiments, the hepatocyte, tissue, or organ metabolizes a lipid,cholesterol, or carbohydrate. In some embodiments, the hepatocyte,tissue, or organ metabolizes a pharmaceutical drug or toxic substance.In some embodiments, the hepatocyte, tissue, or organ uptakes ammonia orbile acid.

In some embodiments, a hepatocyte herein has comparable phenotypic(e.g., immunophenotypic) properties as a primary hepatocyte. In someembodiments, a hepatocyte herein has comparable morphologic propertiesas a primary hepatocyte. In some embodiments, a hepatocyte herein hascomparable functional properties as a primary hepatocyte.

In some embodiments, a hepatocyte, tissue, or organ disclosed herein hasone or more functions of: synthesis of fatty acids, triglycerides,cholesterol, bile salts, or phospholipids; detoxification, modification,and excretion of exogenous or endogenous compounds (e.g., drug,insecticide, steroid, ammonia, heavy metal, or toxin); carbohydratemetabolism; synthesis of proteins (e.g., serum albumin, fibrinogen,lipoprotein, apoprotein, ceruloplasmin, transferrin, complement, orglycoprotein); protein storage; or formation or secretion of bile. Insome instances, a hepatocyte can be a hepatic progenitor cell (e.g.,hepatocyte-like cell) or a hepatocyte derived from a stem cell; ahepatic stem cell; or a primary hepatocyte (e.g., are or comparable tofreshly isolated or uncultured, cryopreserved hepatocytes obtained froma liver).

In some embodiments, a trophoblast stem cell disclosed herein is derivedfrom an ectopic pregnancy mass (e.g., tubal). In some embodiments, themethod of isolating a trophoblast stem cell herein comprises the stepsof: obtaining trophoblastic villi from an ectopic pregnancy mass (e.g.,tubal); collecting cells from the trophoblastic villi; and culturing thecollected cells in a culture medium to obtain the isolated trophoblaststem cell. In some embodiments, the method further comprises cutting thetrophoblastic villi into pieces. In some embodiments, the method furthercomprises treating the trophoblastic villi with an enzyme. In someembodiments, the human trophoblast stem cell is genetically modified tointroduce a mutation into the cell. In some embodiments, the pregnantmass is obtained in an unruptured manner. In some embodiments, thepregnant mass is at a gestational age of no older than 7 or 8 weeks. Insome embodiments, the culture medium is free of a feeder layer. In someembodiments, the method further comprises the steps of: formingembryonic bodies (EBs) in the culture medium; treating the EBs with anenzyme; and collecting cells from the enzyme-treated EBs to obtain theisolated human trophoblast stem cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F illustrate immunoreactive markers of DE lineages. FIGS.1A-1D show immunocytochemistry to detect FOXA2 and SOX17 (FIG. 1A), GSC(FIG. 1B), and MIXL1 (FIG. 1C) at 4 hr of bFGF induction compared to thecontrol in hTS cells. FIG. 1D shows western blots in time course ofDE-related transcription factors including GSC, Brachyury, MIXL1, SOX17,and FOXA2 at initial 8 hr of bFGF induction. Error bars indicatestandard deviation (SD) of mean. N=3, *: p<0.05 as statisticsignificant. FIG. 1E shows colocalization of hepatocyte-associatedmarkers showing AFP and albumin (upper panel) and ABCC2 and BSEP (lowerpanel) at day-4 after induction. FIG. 1F shows a two-step regimen forhepatocyte-like cell differentiation showing various hepatocyte-specificmarkers by Western blotting assay in hTS cells. α-tubulin as loadingcontrol.

FIGS. 2A to 2M illustrate regulatory Molecular Mechanisms for DESpecification. FIG. 2A indicates miR-124a analysis (ChIP-qPCR) whichidentifies that CREB1 targets at three sites of promoter of miR-124amRNA (SEQ ID NOS 107-109, respectively, in order of appearance). Aschematic drawing of the consensus miR-124a binding sites (upper panel).C: as no-antibody control. Error bars indicate SD of 3 replicates. FIG.2B illustrates that knockdown of CREB1 reduces the bFGF-induced miR-124aexpression by immunoblotting assay. Error bars indicate SD of 3replicates. *: p<0.05. FIG. 2C shows bFGF induced expression ofphosphor(p)-CREB1 and miRNA-124a during 8 hr induction by qPCR assay.Error bars indicate SD of 4 replicates. FIG. 2D shows ChIP-qPCR of Smad4which identifies that miR-124a represses Smad4 expression via targetingtwo sites of the promoter by luciferase reporter assay (SEQ ID NOS110-112, respectively, in order of appearance). The schematic drawing ofthe consensus miR-124a binding sites (upper panel). Empty vector ascontrol; pSmad4 indicating Smad4 plasmid, Error bars indicate SD of 3replicates. *: p<0.05. FIG. 2E shows the effects of bFGF, miR-124a, andanti-miR-124a antibody on the expression of DE-related transcriptionfactors by Western blots. β-actin was used as loading control. FIG. 2Fshows the knockdown of Smad4 using shRNAs represses expression of Smad4and MIXL1 by Western blots. β-actin was used as loading control. FIG. 2Gand FIG. 2H show ChIP-qPCR assays which identify the inhibitory GSK3β(SEQ ID NO: 113) by miR-124a (SEQ ID NO: 114) by luciferase reportassays at 4 hr induction (FIG. 2G); while qPCR assay showed aninhibitory FOXA2 by β-catenin (FIG. 2H). A schematic drawing of theconsensus miR-124a binding sites (G, upper panel). Empty vector ascontrol; pGSK3β indicating GSK3β plasmid, Error bars indicate SD of 3replicates. *: p<0.05. FIG. 2I shows ChIP-qPCR assay which identifiesthe inhibitory CDX2 (SEQ ID NO: 115) by miR-124a (SEQ ID NO: 116) byluciferase reporter assay. A schematic drawing of the consensus miR-124abinding sites (upper panel). Empty vector as control; pCDX2 indicatingCDX2 plasmid, Error bars indicate SD of 3 replicates. *: p<0.05. FIG. 2Jshows imaging which revealed a reciprocal inhibitory mechanism betweenCDX2 and OCT4 at 4 hr bFGF induction in hTS cells. FIG. 2K shows westernblots in timeline of pluripotent transcription factors CDX2 and OCT4(upper panel) as well as NANOG, and SOX2 (lower panel) during DEdifferentiation. Error bars indicate SD of 3 replicates. *: p<0.05. FIG.2L shows ChIP-qPCR assay which identifies binding of OCT4 at the twosites of the promoter of SOX/7 gene. Error bars indicate SD of 3replicates. FIG. 2M shows a schematic illustration of bFGF induction inthe differentiation of hTS cells towards DE lineages.

FIGS. 3A to 3C illustrate morphogenesis of hepatocyte-like cells. FIG.3A shows morphological changes of cells the differentiation, forming aplate-like tissue structure at day 6-8 day of induction. FIG. 3B showselectron micrographs revealed the infrastructure: largecytoplasm/nucleus ratio, plenty of mitochondria (m), Golgi apparatus(Gi), well-organized endoplasmic reticulum (RER), the junctionalcomplexes (white arrow) to form bile canaliculus lumen (Cn), and thejunctional complexes (double arrows) seal off the space from theremaining extracellular space, lamelleted inculsion at cytoplasm, anddesmosome junction (arrow). FIG. 3C shows immunohistochemistry of thehepatic plate-like tissue showing immunoireactive cell membrane markersof hepatocytes: CXCR4, CX32, BSEP, and MRP2 (ABCC2) and cytoplasmicmarkers: Betatrophin, HNF4α, Albumin, AFP, CYP2B6, APOF, CPS1, and ADH1.

FIGS. 4A to 4D illustrate liver functions of the differentiatedhepatocyte-like cells. FIG. 4A shows differentiated hepatocyte-likecells (4 days) exhibiting protein secreting capacity in the medium suchas albumin and urea measured by an automatic analyzer (Hitachi 7080;Tokyo, Japan). Error bars indicate SD of 3 replicates. **: p<0.01. FIG.4B and FIG. 4C show a LDL uptake assay which illustrates immunoreactiveLDL receptor (LDLR, middle panel) and LDL staining (left panel) (FIG.4B) and Oil-O-Red test showing fat droplets (FIG. 4C) in thedifferentiated hepatocyte-like cells. FIG. 4D shows glycogen storagetest identifies the presence of glycogen in the cells by periodicacid-Schiff (PAS) staining evidenced by diastase to digest glycogen(upper pane) and confirmed by fluorescent PAS staining (lower panel) inthe hepatocyte-like cells (left 3 panel) and the hepatic plate-liketissue (right panel).

FIGS. 5A to 5I illustrate a variety of CYP 450 enzyme activities in thedifferentiated hepatocyte-like cells. Phase I-II CYP450 enzyme activityis estimated by drug-drug interaction and detoxification tests (inducer,inhibitor), including CYP1A2 (FIG. 5A), CYP2B6 (FIG. 5B), CYP2C8 (FIG.5C), CYP2C9 (FIG. 5D), CYP2C19 (FIG. 5E), CYP2D6 (FIG. 5F), CYP2E1 (FIG.5G), CYP3A4 (FIG. 5H), and CYP 7A1 (FIG. 5I). Inducer and inhibitor usedas (rifampin, Rif and ciprofloxacin, Cip), (phenobarbital, phen andCip), (Rif and gemfibrozil, Gem), (Rif and Gem), (Rif and ticlopidine,Tico), (Rif as inducer only), (Rif as inducer only), (Rif, anditraconazole, Itra), and (THA, 2,4,6-trihydroxyacetophenone and CDCA,chenodeoxycholic acid), respectively. hTS indicating hTS cells, hTHLindicating human trophoblast-derived hepatocyte-like cells; and Huh7indicating human hepatoma Huh7 cells.

FIG. 6 illustrates a table showing biomarker expression (e.g. mRNAs)during the different stages of a hepatocyte differentiation.

FIG. 7 illustrates the cellular processes of DE formation by TissueFAXanalysis. bFGF (10 ng/mL) was used to induce differentiation of hTScells to mesendoderm indicated by upregulation of MIXL1 (black).Subsequently, the mesendoderm differentiated into DE lineage at about 4hours induction, expressing a downregulation of MIXL1 (grey). nindicates the total number of cells counted.

FIG. 8A to FIG. 8D illustrate expression level analysis of biomarkersdescribed herein. FIG. 8A illustrates that FGFR inhibitor (PD166866)blocks bFGF-induced PI3K with β-actin as a loading control. FIG. 8Billustrates that PI3K siRNA inhibits the expression of PI3K and p-AKT.Cells transfected with non-specific shRNA were used as control. β-actinwas used as a loading control. FIG. 8C shows that siRNAs against AKTsubunits inhibits the bFGF-induced expression of p-AKT and p-CREB1.Cells transfected with non-specific shRNA were used as control. β-actinwas used as a loading control. FIG. 8D shows that AKT interacts directlyto CREB1 by IP assay.

FIG. 9A to FIG. 9F illustrate the genetic fluctuation profiles ofhepatic development-associated 31 genes after induction by qPCR analysisin hTS cells.

FIGS. 10A to 10D and 10E to 10J illustrate immunoreactive markers duringDE formation. (10A) Western blot analysis in time course ofrepresentative markers of primitive streak and DE markers at the initialinduction (8 hr). Data indicating mean±SD, n=3, *: p<0.05 as statisticsignificant. (10B) immunocytochemistry of Foxa2 and Sox17 (left panel),Gsc (middle panel), and Mixl1 (right panel) at 4 hr of bFGF induction.(10C) Identification of CREB1 in targeting at three sites of promoter(SEQ ID NOS 107-109, respectively, in order of appearance) (upper panel)in miR-124a to increase its levels by ChIP-qPCR. C: as control. Datarepresenting mean±SD, n=3, *: p<0.05 as statistic significant. (10D)bFGF induces a parallel expression between phosph(p)-CREB1 andmiRNA-124a by qPCR assay. Data indicating mean±SD, n=4, *: p<0.05 asstatistic significant. (10E) A shifting mean Mixl1 intensity in cellswithin 4 hr induction by TissueFAX analysis. Blank area as control, bluearea as mesendoderm stage, red area as DE stage. (10F) FGFR inhibitor(PD 166866) blocks the bFGF-induced PI3K by Western blotting. β-actinwas used as loading control. (10G) PI3K siRNA inhibits expression ofPI3K and p-Akt. Cells transfected with non-specific shRNA are used ascontrol. β-actin was used as loading control. (10H) siRNAs against Aktsubunits inhibits the bFGF-induced expressions of p-Akt and p-CREB1.Cells transfected with non-specific shRNA are used as control. β-actinwas used as loading control. (10I) Akt interacts directly to CREB1 by IPassay. (10J) bFGF-induced miR-124a is inhibited by using CREB1 shRNAs.Data representing mean±SD, n=3, *: p<0.05 as statistic significant.

FIGS. 11A to 11I and 11J to 11K illustrate molecular mechanisms for DEspecification. (11A, 11B, 11C) Luciferase reporter assays of miR-124arepressing the expressions of Smad4 plasmid (pSmad4) (A), pGSK3β (B),and pCdx2 (C) via targeting the promoter(s) of gene (upper panel). Emptyvector: control, Data indicating mean±SD, n=3, *: p<0.05 as statisticsignificant. FIG. 11A discloses SEQ ID NOS 110-112, respectively, inorder of appearance, and FIG. 11C discloses SEQ ID NOS 115-116,respectively, in order of appearance. (11D) β-catenin binds to theregion (−2.1 kb) of promoter in Foxa2 gene over time by ChIP-qPCR assay.Error bars indicate SD of 3 replicates. (11E) Foxa2 targets the promoterof Betatrophin by ChIP assay, showing production of betatrophin at 12 hrinduction (arrow). Input: whole cells as positive control. IgG asnegative control. FIG. 11E discloses SEQ ID NOS 113-114, respectively,in order of appearance. (11F) Expression of various transcriptionfactors in response to miR-124a and anti-miR-124a antibody at 4 hr ofbFGF induction by Western blot analysis. β-actin: loading control. (11G)A reciprocal inhibitory function between Cdx2 (green) and Oct4 (red) at4 hr of bFGF induction immunocytochemically. (11H) Oct4 binds to tworegions (−1 and −1.8 kb) of promoter in Sox17 gene at 2 hr of bFGFinduction by ChIP-qPCR assay. Error bars indicate SD of 3 replicates.(11I) Schematic illustration of molecular regulation in DEdifferentiation of hTS cells. (11J) Smad4 shRNAs inhibit expression ofMixl1 by immunoblotting assay. (11K) Expression of Oct4, Cdx2, Nanog,and Sox2 in time course during DE formation. Data indicating mean±SD,n=3, *: p<0.05.

FIGS. 12A to 12D and 12E to 12F illustrate biological characteristics ofhepatocyte-like cells. (12A) H&E staining of the crescent cell mass(left panel, insert). Numerous clustered cells distributed irregularlyat the outer peripheral layer containing abundant small embryonicprogenitor-like cells with condensed nuclei (right lower). Columnartree-like ECMs along with hepatocyte-like cell linings radiating fromthe peripheral layer to the central areas (right upper). (12B)Hepatocyte-like cells in the CCl4-damaged liver tissues, showingimmunoreactive stem-121-positive cells (left upper), C-kit positivecells (right upper, arrow), CK19 (left lower, arrow), and CK18 (rightlower, arrow). (12C) A variety of specific immunoreactive markers duringliver development observed in the crescent cell mass histologically.Cellular surface markers make up polygonal shape of hepatocyte seen insmall insert. (12D) Representative electron micrographs showing: a largecytoplasm/nucleus ratio, plenty of mitochondria (m), endoplasmicreticulum (rer), lipid droplets, space of Disse (SD), extracellularmatrix (ecm), nucleus (n), and sinusoid in upper micrograph, glycogen(gly) storage with rosette formation (red circle) in lower leftmicrograph, and lower right micrographs showing bile canaliculus lumen(bc) and junctional complex (upper) and tight junction (lower). (12E)Immunoreactive albumin (ALB) and AFP expressed in the hepatocyte-likecells. (12F) Morphological changes during hTS cells differentiation tohepatocyte-like cells in time course.

FIGS. 13A, 13B, and 13C illustrate secretomics in hepatocyte-like cellculture medium. (13A) Proteomic analysis of culture medium before cellculture (as control, left panel) and after 5-day cell culture (rightpanel) revealing a new formation of protein (designated as No. 413,circle). (13B) No immunoreactive TGFβ1, collagen IV (COL4), andfibronectin (FN) expressed in hTS cells before induction (upper panels)and after induction for 5-days, coexpression of them distribute ascolumnar ECMs between hepatocyte-like cells in 3-D structure (lowerpanels). (13C) Mascot MS/MS ions search system analysis 20151001 LiP413, transforming growth factor-beta-induced protein ig-h3 precursor[Homo sapiens] (SEQ ID NO: 117).

FIGS. 14A to 14E illustrate functional Characteristics ofHepatocyte-Like Cells. (14A) HepatoHepatocyte-like cells secrete albumin(left) and urea (right; by stimulation of 5 mM ammonium chloride for1-day) into the cultured medium by automatic analyzer (Hitachi 7080;Tokyo, Japan). Error bars indicate SD of 3 replicates. **: p<0.01. (14B)LDL uptake assay shows immunoreactive LDL (red, left), LDL receptor(LDLR, green, middle), and their emerged image (right) in the cells.(14C) Oil-O-Red test shows fat droplets (red) in the cells. (14D)Glycogen storage test identifies the presence of glycogen (pink and red)by periodic acid-Schiff (PAS) staining using diastase treatment (upperpanel) and also by fluorescent PAS staining (lower panel). (14E) Avariety of phase I-II CYP 450 enzyme activity in response to inducer(green) and inhibitor (pink) at 24 hr treatment in hepatocyte-like cellsby qPCR analysis. Abbreviations: Rif, rifampin; Cip, ciprofloxacin;Itra, itraconazole; Phen, phenobarbital; Gem, gemfibrozil; THA,2,4,6-trihydroxyacetophenone; CDCA, chenodeoxycholic acid; and Tico,ticlopidine.

FIGS. 15A to 15F illustrate responsiveness of intravenoustransplantation by hepatocyte-like cells (15A) Serum levels of AST andALT are higher in cell therapy group (CCl4+cells; n=8) than controlgroup (CCl₄ only; n=8) over time. Data represent mean±s.e.m., Studenttest: *: p<0.01. (15B) Expression of immunoreactive stem-121 in hTScells (upper) and in hepatocyte-like cells resided in liver tissues(lower). (15C) Stem-121-positive hepatocytes in the CCl₄-damaged livertissues expressing characteristics of cellular degeneration (insert). PTindicating portal triad. (15D) Coexpression of immunoreactive Stem-121and HLA-G in the implanted hepatocyte-like cells. Bar scale: 20 (15E,15F) Distribution of immunoreactive CD4⁺Foxp3⁺ Treg cells among theCCl4-damaged hepatocytes immunocytochemistry (15E) and immunoreactiveCD4⁺ cells (red) around a central vein immunohistochemistry (15F).

DETAILED DESCRIPTION

Disclosed herein are methods, compositions, cells, manufacture process,and kits for generating an induced hepatocyte from a trophoblast stemcell. In some embodiments, described herein is a method of inducing atrophoblast stem (TS) cell to differentiate into an induced hepatocytein vitro, that comprises (a) contacting the trophoblast stem cell in aconditioned medium comprising a fibroblast growth factor (FGF), asteroid, and a cytokine; and (b) incubating the cell for sufficient timeto induce differentiation of the trophoblast stem cell into an inducedhepatocyte.

Also described herein is an isolated induced hepatocyte derived from atrophoblast stem cell, wherein the isolated induced hepatocyte comprisesan elevated level of expression of one or more biomarkers comprisingC—X—C chemokine receptor type 4 (CXCR4), Forkhead box protein A2(FOXA2), SRY-box 17 (SOX17), hexosaminidase A (alpha polypeptide)(HHEX), bile salt export pump (BSEP), transthyretin (TTR), albumin(ALB), tyrosine aminotransferase (TAT), cytochrome P450 7A1 (CYP7A1),glucose-6-phosphatase (G6PC), serpin peptidase inhibitor clade A(alpha-1 antiproteinase, antitrypsin) member 1 (SERPINA1), ATP-bindingcassette sub-family C (ABCC2), CCAAT-enhancer-binding protein beta(C/EBPβ), hepatocyte nuclear factor 1-alpha (HNF1α), hepatocyte nuclearfactor 4-alpha (HNF4α), alpha-1-fetoprotein (AFP), keratin 8 (KRT8),phosphoenolpyruvate carboxykinase 2 mitochondrial (PCK2), cytochromeP450 2B6 (CYP2B6), glycogen synthase 2 (GYS2), hepatocyte nuclear factor6 (HNF6), carbamoyl-phosphate synthase 1 mitochondrial (CPS1), alcoholdehydrogenase 1C (class I) gamma polypeptide (ADH1C), connexin 32(CX32), cytochrome P450 3A4 (CYP3A4), prospero homeobox 1 (PROX1),tryptophan 2,3-dioxygenase (TDO2), apolipoprotein F (APOF), keratin 18(KRT18), keratin 19 (KRT19), or chromosome 19 open reading frame 80(angiopoietin-like protein 8, hepatocellular carcinoma-associated geneTD26, lipasin) (Betatrophin).

Further described herein is a method of screening a compound for use intreatment or prevention of a disease or disorder, which comprises (a)contacting an isolated induced hepatocyte herein with the compound; and(b) detecting the expression level of a biomarker in the isolatedinduced hepatocyte.

Described herein, in addition, are compositions (e.g. pharmaceuticalcompositions) that comprises an isolated induced hepatocyte disclosedherein, manufacture process for generating a composition (e.g.pharmaceutical composition) that comprises an isolated inducedhepatocyte disclosed herein, and methods of treating a disease ordisorder (e.g. a liver-associated disease or disorder) with an isolatedinduced hepatocyte disclosed herein or a composition that comprises anisolated induced hepatocyte disclosed herein.

In some aspects, disclosed herein is a highly efficient generation ofhepatocyte-like cells from ectopic pregnancy-derived human trophoblast(hTS) stem cells, exhibiting molecular, genetic, and biologicalcharacteristics resemblance to primary hepatocytes in liver development.In some embodiments, disclosed herein is a mechanism of microRNA-124acontrolling definitive endoderm formation during differentiation. Insome embodiments, hepatocyte-like cells can construct a 3-D liverplate-like structure in cell culture, expressing HLA-G and secretingTGFβ1 to maintain CD4⁺Foxp3⁺ Treg cells in liver tissues for immunetolerance after intravenous implantation. In some embodiments, the cellsherein assist and promote liver regeneration in rat model ofCCl₄-induced acute liver failure. In some embodiments, hTS cell-derivedhepatocyte-like cells herein can be applied in the urgent management ofliver failure or in regenerative medicine. In some embodiments,disclosed herein is efficient two-step differentiation of hTS cells tofunctional hepatocytes within a week (e.g., 4-6 days). In someembodiments, miR-124a controls DE formation during hepatogenesis. Insome embodiments, disclosed herein are hepatocyte-like cells thatconstruct 3-D tissue structure with biological functions mimickingprimary hepatocytes.

In some aspects, disclosed herein is intravenous infusion ofhepatocyte-like cells can homed to the CCl₄-damaged liver tissues topromote liver regeneration in rat animal model. In some embodiments,disclosed herein are both hTS cells and its derivative hepatocyte-likecells express HLA-G to obtain immune tolerance after transplantation. Insome embodiments, disclosed herein are homing hepatocyte-like cellssecret TGFβ1 to assist the construction of new ECMs after injury via theformation of fibronectin and collagen. In some embodiments,Hepatocyte-like cell-secreted TGFβ1 resulting in the bone marrow'sfibrocytes migration to liver, activates hepatic stellate cells forliver regeneration and maintains CD4⁺Foxp3⁺ Treg cells in liver tissuesfor immune tolerance. In some embodiments, basic fibroblast growthfactor (bFGF) alone induces activation of microRNA (miRNA)-124a toconsequently control the DE specification in early differentiation. Insome embodiments, with certain conditions, DE gives rise to hepaticendoderm followed by hepatoblasts and eventually differentiates tofetal/adult hepatocyte-like cells, bearing similar genetic, molecularand biological characteristics to primary human hepatocytes.

In some aspects, hepatocyte-like cells enable to build athree-dimensional (3-D) tissue structure in vitro and intravenousinfusion of such cells results in hepatic homing and protects the liverfrom damage. In some embodiments, a tissue-culture media compositionused herein comprises about serum and culture medium. In someembodiments, the culture medium is Synthetic Oviductal Fluid (SOF),Modified Eagle's Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), RPMI 1640, F-12, IMDM, Alpha Medium, or McCoy's Medium. In someembodiments, the serum is allogeneic serum, autologous serum, orxenogeneic serum. In some embodiments, hTS cells are cultured with acombination of fibroblast growth factor (e.g., bFGF), steroid (e.g.,dexamethasone), cytokine (e.g., oncoststin M), bone morphogeneticprotein (e.g., BMP4), and hepatic growth factor (HGF) after DE formation(e.g., 8 hr). In some embodiments, the resulting cells form dispersedfibroblast-like cells. In some embodiments, the resulting cellsgradually aggregate to form a crescent cell mass. In some embodiments,two distinct peripheral and central compartments construct a3-dimensional (3D) tissue structure. In some embodiments, in theperipheral part, numerous clustered small cells distribute irregularlyamong the extracellular matrix (ECM) beyond the basement membrane. Insome embodiments, cells have condensed nuclei, frequently eccentriclocated, and abundant granular and vacuoles in the eosinophiliccytoplasm similar to the embryonic stem/progenitor cells. In someembodiments, in the central part, many independent columnar ECMs, bycell linings at both sides, distribute from the basal towards thecentral areas. These cells contain abundant eosinophilic cytoplasm anddisperse chromatin in the single round nucleus with one or two prominentnucleoli mimicking the phenotypic hepatocytes. In some embodiments,several binucleate cells can form, similar to hepatic plates in humanliver.

In some embodiments, the hepatocyte or hepatocyte-like cells hereinexhibit specific marker(s) of: i) human cytoplasmic marker stem 121™ forhuman cells, mast/stem cell growth factor receptor C-kit for liverintrinsic stem cells, CK19 for cholangiocytes, and CK18 for hepatocytes;and ii) albumin (ALB), α-fetoprotein (AFP), Betatrophin, ADH1, APOF,CPS1, GATA4, CYP1A1, and CYP2B6 in the cytoplasm for hepatocytesimmunohistochemically. In some embodiments, a subset of surface markersincluding ASGR1, CXCR4, BSEP, MRP2, and Cx32 construct a polygonal cellshape similar to the primary human hepatocyte, e.g., a similarultrastructure to primary hepatocyte, including a large cytoplasm tonucleus ratio, plenty of mitochondoria, well-organized endoplasmicreticulum, tight junction, numerous lipid vacuoles, glycogen storage,enlarged lumen of the bile canaliculus with junctional complexes, andmultiplex ECMs.

Among 9 newly upregulated, secreted proteins in the cell-culturedmedium, protein (no. 413) significantly predicts, by 46% of peptidesequences matched, to be the transforming growth factor-β(TGF-β)-induced protein ig-h3 precursor (TGFβ1) by Mascot MS/MS ionssearch system (ESI-QUAD-TOF, Bruker Impact HD, Matrix Science, USA).TGFβ1 is a major fibrogenic, multifunctional cytokine, acting as bothautocrine and paracrine manner to enhance fibronectin and collagenformation in hepatic stellate cells (HSCs). In some embodiments, TGFβ1is expressed in ECM. In some embodiments, TGFβ1, fibronectin, andcollagen IV are co-expressed in the ECMs. In some embodiments, TGFβ1,fibronectin, and collagen IV constitute, at least partly, the scaffoldof ECMs in the 3-D tissue structure of hepatocyte-like cells that maysupport proliferation and differentiation of hepatocytes in the hepaticplates.

In some aspects, hepatocyte-like cells can be efficiently generated frompluripotent hTS cells through a series of cellular processes, includingthe primitive streak, DE formation, hepatic endoderm, hepatoblasts, andultimately hepatocyte-like cells. The onset of primitive streakdifferentiation can be verified at the initial induction (e.g., 30 min)by upregulation of GSC, Brachyury, and Mixl1. The immediately decreasedMixl1 can perform an impact on the endoderm potential of the mesendodermprogenitors. Moreover, Sox7-expressing cells can be originally presentat the extra-embryonic endoderm but not at the DE lineages. Asdevelopment progresses, the apparent upregulation of Sox17 and Foxa2 aswell as downregulation of Mixl1 can define the formation of DE, in whichOct4 play a main role in the maintenance of pluripotency distinct fromNanog in the hES cell- or iPS cell-derived DE.

In some aspects, a transient elevation of miR-124a can negativelymodulate multiple gene expressions post-transcriptionally via binding tothe targeted mRNAs, typically in the 3′UTR to control DE specification.There can be presence of functionally silenced miR-124a in the earlyhepatic differentiation. Wherein, the downregulated miR-124a afterpeaking at 4 hr induction bears a resemblance to the scenario when cellmigration begins in gastrulation to form three embryonic germ layers inhES cells.

In some aspects, the initiation of hepatic lineage differentiationfollowing DE specification can be achieved by a combination of bFGF,dexamethasone, oncoststin M, BMP4, and HGF The stage-specific geneprofiles can indicate a committed step in the hepatic specified endoderm(Table 3, second column). For example, expression of α-fetoprotein (AFP)expression suggests the initial differentiation of hepatic endoderm andboth C/EBPβ and Hnf4α control initial liver-specific activity in theurea cycle. Expression of α-1-antitrypsin (SERPINA1) can protect cellsfrom damage and promotes metabolic activity of enzymes such as CYP7A1,CYP3A4, and CYP2B6. These facts represent that hepatic endoderm iscapable of metabolism of cholesterol, drug, and toxin at the earlydifferentiation. Since Sox17 directly induces zinc finger protein 202(ZFP202) to suppress the master hepatic gene regulator Hnf4α, thereby,withdrawal of Sox17 facilitated the initiation of Hnf4α expression afterDE stage, which, in turn, to characterize the specification of hepaticprogenitor cells, controlling hepatocyte cell fate. A sustained Foxa2can be responsible for the consequent expressions of albumin, AFP,mitochondrial protein TAT, and betatrophin; while betatrophin expressionreflects the early capacity in the promotion of β cell proliferation andlipid metabolism at the early hepatic differentiation.

In some aspects, as differentiation proceeds, numerous hepatic markersbegin to emerge, including PROX1, G6PC, Hnf1α, ABCC2, and TDO2 as wellas cytokeratins such as CK8, CK18, and CK19. PROX1, for example, isrequired for hepatoblastic migration and its ablation in hepatoblastscauses defective hepatocyte specification and promotes biliary cellcommitment. CK8 is an intermediate filament protein to polymerize withCK18 forming a component of the epithelial cytoskeleton and acts as aplasminogen receptor. Hepatoblasts in between 2 and 4 daysdifferentiation can express cholangiocyte marker CK19 and hepaticprogenitor markers CK8 and CK18 (Table 3), mimicking the bipotentialcapacity in differentiation to biliary epithelial cells and hepatocytes,respectively. Expression of the hepatocyte-enriched transcription factorcluster, including Foxa2, Hnf1α, Hnf4α, and Hnf6, can represent amilestone of the hepatoblastic differentiation, directing theparenchymal hepatoblasts into hepatocytes and promoting hepatocytematuration. For metabolism, expression of hepatobiliary excretiontransporter MRP2 (ABCC2) and hepatic gap junction protein Cx32 canfacilitate the transport of various molecules across cellular membranes.An upregulation of HHEX, however, can implicate the presence ofhematopoietic capacity in the hepatoblasts.

In some aspects, implanted hTS cell-derived hepatocyte-like cells cansurvive to reach a subject's liver after intravenous transplantation. Insome embodiments, the cells herein have a homing instinct. In someembodiments, these cells can express HLA-G, a nonclassical HLA class Imolecule, which, membrane-bound or soluble, strongly acts on differentimmune cell types (NK, T, B, monocytes/dendritic cells) to inhibit bothinnate and adaptive immunity through the interaction with the inhibitoryreceptors that are expressed at the surface of immune cells.Additionally, these hepatocyte-like cells can enable to recruitCD4⁺Foxp3⁺ regulatory T (Treg) cell population postimplantation,contributing to the generation of an immunosuppressive environment bythe inhibition of proinflammatory T cells and the induction of T cellswith a regulatory. In some embodiments, hTS cell-derived hepatocyte-likecells possess immune privilege.

In some aspects, provided herein are compositions and methods fortransplanting hepatocyte or hepatocyte-like cells to subjects. In someembodiments, the subject is injected by hTS cell-derived hepatocytes(e.g., intravenously, intramuscularly, transdermally, endoscopicretrograde injection, or intraperitoneally). In some embodiments, thesubject is not treated with an immunosuppressive agent prior to thetransplanting. In some embodiments, the method further comprisestreating the patient with an immunosuppressive agent, e.g., FK-506,cyclosporin, or GAD65 antibodies.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range, e.g., ±15% of a referenced numeral value.About also includes the exact amount. Hence “about 5 μL” means “about 5μL” and also “5 μL.” Generally, the term “about” includes an amount thatwould be expected to be within experimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Overview

Liver possesses a dynamic range of functions including detoxification,protein synthesis, protein storage, and production of biochemicalcomponents necessary for digestion. It comprises two major types ofcells, parenchymal and non-parenchymal cells. Parenchymal cells make upabout 80% of the liver volume and are also referred to as hepatocytes.Non-parenchymal cells contribute to about 6.5% of liver volume, butconstitute to about 40% of the total number of liver cells. In someinstances, non-parenchymal cells comprise sinusoidal hepatic endothelialcells, Kupffer cells, and hepatic stellate cells.

Hepatocytes

Hepatocytes, or parenchymal cells, are responsible for the function ofthe liver. In some instances, hepatocytes are involved in proteinsynthesis, protein storage, synthesis of cholesterol, bile salts andphospholipids, formation and secretion of bile, carbohydrate metabolism,and detoxification, modification, and excretion of exogenous andendogenous substances.

In some instances, proteins synthesized from the hepatocytes includemajor plasma proteins such as human serum albumin, soluble plasmafibronectin, α-fetoprotein, C-reactive protein, and several globulins;proteins involved in hemostasis and fibrinolysis such as coagulationfactors involved in the coagulation cascade, α2-macroglobulin,α1-antitrypsin, antithrombin III, protein S, protein C, plasminogen,α2-antiplasmin, and complement component 3; carrier proteins such asalbumin, ceruloplasmin, transcortin, haptoglobin, hemopexin, IGF bindingrotein, major urinary proteins, retinol binding protein, sexhormone-binding globulin, transthyretin, transferrin, and VitaminD-binding protein; hormones such as insulin-like growth factor 1,thrombopoietin, hepcidin, and betatrophin; prohormones such asangiotensinogen; and apolipoproteins.

In addition to formation, breakdown, and interconversion ofcarbohydrates, in some instances, carbohydrate metabolism also involvesgluconeogenesis, glycogenolysis, and glycogenesis. Gluconeogenesis isthe synthesis of glucose from certain amino acids, lactate or glycerol.Glycogenolysis is the breakdown of glycogen into glucose. Glycogenesisis the formation of glycogen from glucose.

In some instances, lipid metabolism within hepatocytes includescholesterol synthesis and lipogenesis, the production of triglyceridesor fats.

In some cases, after injuries such as tissue damage or tissue loss,hepatocytes can re-enter the cell cycle leading to proliferation andsubsequent regeneration of the injured portion, such as the damaged orlost tissue. In some instances after the removal of liver tissue, theremaining hepatocytes undergo at least one, two, three, or more roundsof DNA synthesis leading to regeneration of the lost tissue mass.

In some embodiments, hepatocytes are utilized for pharmaceuticalresearch. In some embodiments, these researches include drug metabolism,enzyme induction, hepatotoxicity, hepatocyte regeneration, andtransplantation.

The term hepatocyte refers to a hepatic cell or hepatic progenitor cellthat has one or more functions of: synthesis of fatty acids,triglycerides, cholesterol, bile salts, or phospholipids;detoxification, modification, and excretion of exogenous or endogenouscompounds (e.g., drug, insecticide, steroid, ammonia, heavy metal, ortoxin); carbohydrate metabolism; synthesis of proteins (e.g., serumalbumin, fibrinogen, lipoprotein, apoprotein, ceruloplasmin,transferrin, complement, or glycoprotein); protein storage; or formationor secretion of bile. In some instances, a hepatocyte can be a hepaticprogenitor cell (e.g., hepatocyte-like cell) or a hepatocyte derivedfrom a stem cell; a hepatic stem cell; or a primary hepatocyte (e.g.,are or comparable to freshly isolated or uncultured, cryopreservedhepatocytes obtained from a liver).

In some embodiments, a hepatic stem cell is a small epithelial celladhesion molecule-expressing (EpCAM-expressing) cell that constitutesabout 0.5%-2.5% of the liver parenchyma. In some embodiments, the stemscell includes, but is not limited to, embryonic stem cell, adult stemcell, inducible pluripotent stem (iPS) cell, parthenogenetic stem cells,or trophoblast stem cell. In some embodiments, the stem cell is a humanstem cell. In some embodiments, the stem cell is a trophoblast stemcell. In some embodiments, the trophoblast stem cell is a humantrophoblast stem cell. In some embodiments, the human trophoblast stemcell is an ectopic pregnancy-derived human trophoblast stem cell. Insome instances, a hepatocyte derived from a stem cell is also referredto as an induced hepatocyte. In some embodiments, an induced hepatocyteis derived from a human trophoblast stem cell. In some embodiments, aninduced hepatocyte is derived from an ectopic pregnancy-derived humantrophoblast stem cell. In some embodiments, an induced hepatocytecomprises a trophoblast stem cell undergoing the process ofdifferentiation into a hepatocyte, and a differentiated trophoblast stemcell. In some embodiments, a trophoblast stem cell undergoing theprocess of differentiation into a hepatocyte is also referred to as animmature induced hepatocyte. In some embodiments, a differentiatedtrophoblast stem cell is also referred to as a mature inducedhepatocyte.

In some embodiments, an induced hepatocyte functions similarly to aprimary hepatocyte. In some embodiments, an induced hepatocyte comprisescellular functions exhibited by a primary hepatocyte. In someembodiments, an induced hepatocyte participates in cellular functionssuch as for example protein synthesis, protein storage, synthesis ofcholesterol, bile salts and phospholipids, formation and secretion ofbile, carbohydrate metabolism, and detoxification, modification, andexcretion of exogenous and endogenous substances, which are observed ina primary hepatocyte. In some embodiments, hepatocytes herein express asubset of surface markers including ASGR1, CXCR4, BSEP, MRP2, and Cx32and construct a polygonal cell shape similar to the primary humanhepatocyte, e.g., a similar ultrastructure to primary hepatocyte,including a large cytoplasm to nucleus ratio, plenty of mitochondoria,well-organized endoplasmic reticulum, tight junction, numerous lipidvacuoles, glycogen storage, enlarged lumen of the bile canaliculus withjunctional complexes, and multiplex ECMs.

In some instances, proteins synthesized from an induced hepatocyteinclude major plasma proteins such as human serum albumin, solubleplasma fibronectin, α-fetoprotein, C-reactive protein, and severalglobulins; proteins involved in hemostasis and fibrinolysis such ascoagulation factors involved in the coagulation cascade,α2-macroglobulin, α1-antitrypsin, antithrombin III, protein S, proteinC, plasminogen, α2-antiplasmin, and complement component 3; carrierproteins such as albumin, ceruloplasmin, transcortin, haptoglobin,hemopexin, IGF binding rotein, major urinary proteins, retinol bindingprotein, sex hormone-binding globulin, transthyretin, transferrin, andVitamin D-binding protein; hormones such as insulin-like growth factor1, thrombopoietin, hepcidin, and betatrophin; prohormones such asangiotensinogen; and apolipoproteins.

In some embodiments, carbohydrate metabolism such as the formation,breakdown, and interconversion of carbohydrates, gluconeogenesis,glycogenolysis, glycogenesis, lipid metabolism including cholesterolsynthesis, and lipogenesis, the production of triglycerides or fats, areobserved in an induced hepatocyte.

In some embodiments, an induced hepatocyte comprises similarultrastructure, or the cellular makeup, as a primary hepatocyte. In someinstances, this is achieved through comparison based on transmissionelectron microscopy images.

In some embodiments, an induced hepatocyte is utilized forpharmaceutical research, such as drug metabolism, enzyme induction,hepatotoxicity, hepatocyte regeneration, and transplantation.

Trophoblast Stem Cells (hTS Cells)

Trophoblast stem cells (TS cells) are precursors of differentiatedplacenta cells. In some instances, a TS cell is derived from ablastocyst polar trophectoderm (TE) or an extraembryonic ectoderm (ExE)cell. In some cases, TS is capable of indefinite proliferation in vitroin an undifferentiated state, and is capable of maintaining thepotential multilineage differentiation capabilities in vitro. In someinstances, a TS cell is a mammalian TS cell. Exemplary mammals includemouse, rat, rabbit, sheep, cow, cat, dog, monkey, ferret, bat, kangaroo,seals, dolphin, and human. In some embodiments, a TS cell is a human TS(hTS) cell.

In some instances, TS cells are obtained from fallopian tubes. Fallopiantubes are the site of fertilization and the common site of ectopicpregnancies, in which biological events such as the distinction betweeninner cell mass (ICM) and trophectoderm and the switch from totipotencyto pluripotency with major epigenetic changes take place. In someinstances, these observations provide support for fallopian tubes as aniche reservoir for harvesting blastocyst-associated stem cells at thepreimplantation stage. Blastocyst is an early-stage preimplantationembryo, and comprises ICM which subsequently forms into the embryo, andan outer layer termed trophoblast which gives rise to the placenta.

In some embodiments, a TS cell is a stem cell used for generation of aprogenitor cell such as for example a hepatocyte. In some embodiments, aTS cell is derived from ectopic pregnancy. In some embodiments, the TScell is a human TS cell. In one embodiment, the human TS cell derivedfrom ectopic pregnancies does not involve the destruction of a humanembryo. In another embodiment, the human TS cell derived from ectopicpregnancies does not involve the destruction of a viable human embryo.In another embodiment, the human TS cell is derived from trophoblasttissue associated with non-viable ectopic pregnancies. In anotherembodiment, the ectopic pregnancy cannot be saved. In anotherembodiment, the ectopic pregnancy would not lead to a viable humanembryo. In another embodiment, the ectopic pregnancy threatens the lifeof the mother. In another embodiment, the ectopic pregnancy is tubal,abdominal, ovarian or cervical.

During normal blastocyst development, ICM contact per se or its deriveddiffusible ‘inducer’ triggers a high rate of cell proliferation in thepolar trophectoderm, leading to cell movement toward the mural regionthroughout the blastocyst stage and can continue even after thedistinction of the trophectoderm from the ICM. The mural trophectodermcells overlaying the ICM are able to retain a ‘cell memory’ of ICM. Atthe beginning of the implantation, the mural cells opposite the ICMcease division because of the mechanical constraints from the uterineendometrium. However, in an ectopic pregnancy in which the embryo islocated within the fallopian tube, constraints do not exist in thefallopian tubes which result in continuing division of polartrophectoderm cells to form extraembryonic ectoderm (ExE) in thestagnated blastocyst. In some instances, the ExE-derived TS cells existfor up to 20 days in a proliferation state. As such, until clinicalintervention occurs, the cellular processes can yield an indefinitenumber of hTS cells in the preimplantation embryos and such cells canretain cell memory from ICM.

In some instances, TS cells possess specific genes of ICM (e.g. OCT4,NANOG, SOX2, FGF4) and trophectoderm (e.g. CDX2, Fgfr-2, Eomes, BMP4),and express components of the three primary germ layers, mesoderm,ectoderm, and endoderm. In some instances, TS cells express embryonicstem (e.g. human embryonic stem) cell-related surface markers such asspecific stage embryonic antigen (SSEA)-1, -3 and -4 and mesenchymalstem cell-related markers (CD 44, CD90, CK7 and Vimentin). In otherinstances, hematopoietic stem cell markers (CD34, CD45, α6-integrin,E-cadherin, and L-selectin) are not expressed.

Methods of Preparation of Induced Hepatocytes

Disclosed herein, in certain embodiments, is a method of inducing atrophoblast stem (TS) cell to differentiate into an induced hepatocytein vitro, which comprises contacting the trophoblast stem cell in aconditioned medium comprising a fibroblast growth factor (FGF), asteroid, and a cytokine; and incubating the cell for a sufficient timeto induce differentiation of the trophoblast stem cell into an inducedhepatocyte. In some embodiments, the TS cell is a human TS (hTS) cell.In some embodiments, the FGF, the steroid, and the cytokine are humanFGF, human steroid, and human cytokine.

In some embodiments, fibroblast growth factors (FGFs) are a family ofgrowth factors involved in angiogenesis, wound healing, embryonicdevelopment, and cellular proliferation and differentiation processes.In some instances, FGFs are heparin-binding proteins and interacts withheparin sulfate proteoglycans. In some instances, there are 22 membersof the FGF family. Exemplary FGFs include: FGF1, FGF2 (also known asbasic FGF or bFGF or FGF-β), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF15/19, FGF20,FGF21, FGF22, and FGF23. In some embodiments, the fibroblast growthfactor is basic fibroblast growth factor (bFGF, also known as FGF2 orFGF-β). In some embodiments, the bFGF is a human bFGF. In someembodiments, the human bFGF is a recombinant human bFGF, or a fragmentthereof.

In some embodiments, FGF is introduced into the cultured medium at aconcentration of between about 0.001 and about 5000 ng/mL, about 0.01and about 500 ng/mL, about 0.1 and about 100 ng/mL, or about 1 and about50 ng/mL.

In some instances, FGF is introduced into the cultured medium at aconcentration of at least 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 100, 200, 300, 400, 500, 1000 ng/mL or more. In someembodiments, FGF is introduced into the cultured medium at aconcentration of at most 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 100, 200, 300, 400, 500, 1000 ng/mL or less.

In some embodiments, bFGF is introduced into the cultured medium at aconcentration of between about 0.001 and about 5000 ng/mL, about 0.01and about 500 ng/mL, about 0.1 and about 100 ng/mL, or about 1 and about50 ng/mL.

In some instances, FGF is introduced into the cultured medium at aconcentration of at least 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 100, 200, 300, 400, 500, 1000 ng/mL or more. In someembodiments, bFGF is introduced into the cultured medium at aconcentration of at most 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 100, 200, 300, 400, 500, 1000 ng/mL or less.

In some embodiments, a fibroblast growth factor is introduced into thecultured medium comprising hTS cells to initiate hTS celldifferentiation event. In some embodiments, the fibroblast growth factoris bFGF. In some embodiments, a steroid and a cytokine are introducedinto the cultured medium after the addition of FGF (e.g. bFGF).

In some embodiments, a steroid is a chemical involved in a wide range ofphysiological processes such as stress response, immune response,regulation of inflammation, carbohydrate metabolism, protein catabolism,blood electrolyte levels, and behaviors. In some instances, steroidsalso include steroid hormones, such as glucocorticoids,mineralocorticoids, androgens, estrogens, and progestogens. In someembodiments, steroids include, but are not limited to, hydrocortisonetypes such as hydrocortisone, hydrocortisone acetate, cortisone acetate,tixocortol pivalate, prednisolone, methylprednisolone, and prednisone;acetonides such as triamcinolone acetonide, triamcinolone alcohol,mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinoloneacetonide, and halcinonide; betamethasone types such as betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, and fluocortolone; halogenated such ashydrocortisone-17-valerate, halometasone, alclometsone dipropionate,betamethasone valerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate, and fluprednidene acetate; labileprodrug esters such as hydrocortisone-17-butyrate,hydrocortisone-17-aceponate, hydrocortisone-17-buteprate, ciclesonide,and prednicarbate. In some embodiments, a steroid is a naturally derivedor chemically modified steroid. In some embodiments, a steroid isdexamethasone, betamethasone, prednisolone, methylprednisolone,triamcinolone acetonide, triamcinolone alcohol, or hydrocortisone. Insome embodiments, a steroid is dexamethasone. As used herein, the term“dexamethasone” refers to dexamethasone and its derivatives. In someembodiments, dexamethasone is utilized for directing an hTS cell todifferentiate into hepatic lineage. In some embodiments, dexamethasoneis utilized in combination with another agent for directing an hTS cellto differentiate into hepatic lineage. In some embodiments, the anotheragent is a cytokine.

In some embodiments, a steroid is introduced into the cultured medium ata concentration of between about 0.001 and about 100 μM, about 0.005 andabout 5 μM, about 0.01 and about 1 μM, or about 0.05 and about 0.5 μM.

In some instances, a steroid is introduced into the cultured medium at aconcentration of at least 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18,0.19, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10 μM, or more. In some embodiments, a steroid is introducedinto the cultured medium at a concentration of at most 0.001, 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM, or less.

In some embodiments, dexamethasone is introduced into the culturedmedium at a concentration of between about 0.001 and about 100 μM, about0.005 and about 5 μM, about 0.01 and about 1 μM, or about 0.05 and about0.5 μM.

In some embodiments, dexamethasone is introduced into the culturedmedium at a concentration of at least 0.001, 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16,0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10 μM, or more. In some embodiments,dexamethasone is introduced into the cultured medium at a concentrationof at most 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10μM, or less.

In some embodiments, a cytokine is a category of small proteins betweenabout 5-20 dKa that are involved in cell signaling. In some instances,cytokines include chemokines, interferons, interleukins, and tumornecrosis factors. Chemokines can play a role as a chemoattractant toguide the migration of cells, and can be classified into foursubfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines includechemokines from the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5(RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCL26, CCL27, and CCL28; the CXC subfamily: CXCL1, CXCL2, CXCL3,CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; the XC subfamily: XCL1 andXCL2; and the CX3C subfamily CX3CL1.

Interferons (IFNs) comprise interferon type I (e.g. IFN-α, IFN-β, IFN-ε,IFN-κ, and IFN-ω), interferon type II (e.g. IFN-γ), and interferon typeIII. In some embodiments, IFN-α is further classified into about 13subtypes including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8,IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

Interleukins are expressed by leukocytes or white blood cells and theypromote the development and differentiation of T and B lymphocytes andhematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33,IL-35, and IL-36.

Tumor necrosis factors (TNFs) are a group of cytokines that modulateapoptosis. In some instances, there are about 19 members within the TNFfamily, including, not limited to, TNFα, lymphotoxin-alpha (LT-alpha),lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L,FASL, 4-1BBL, OX40L, and TNF-related apoptosis inducing ligand (TRAIL).

In some embodiments, dexamethasone is utilized in combination with acytokine for directing an hTS cell to differentiate into hepaticlineage. In some instances, the cytokine is a chemokine, an interferon,an interleukins, or a tumor necrosis factor. In some instances, thecytokine is an interleukin. In some embodiments, dexamethasone isutilized in combination with an interleukin for directing an hTS cell todifferentiate into hepatic lineage. In some embodiments, the interleukinis IL-6. In some instances, IL-6 is further grouped with additionalcytokines based on its interaction through the Gp130 receptor sub-unit.In some instances, additional members of the IL-6 group includeoncostatin M (OSM), IL-11, Ciliary neurotropic factor (CNTF),Cardiotrophin-1 (CT-1), Cardiotrophin-like cytokine (CLC), and Leukaemiainhibitory factor (LIF). In some embodiments, dexamethasone is utilizedin combination with IL-6 for directing an hTS cell to differentiate intohepatic lineage. In some embodiments, dexamethasone is utilized incombination with a member of the IL-6 group for directing an hTS cell todifferentiate into hepatic lineage. In some embodiments, dexamethasoneis utilized in combination with OSM, IL-11, CNTF, CT-1, CLC, or LIF fordirecting an hTS cell to differentiate into hepatic lineage. In someembodiments, dexamethasone is utilized in combination with OSM fordirecting an hTS cell to differentiate into hepatic lineage. In someembodiments, the IL-6 group of cytokines is human IL-6 cytokines ortheir fragments thereof. In some embodiments, OSM is a human OSM. Insome embodiments, the human OSM is a recombinant human OSM, or itsfragments thereof.

In some embodiments, a cytokine is introduced into the cultured mediumat a concentration of between about 0.001 and about 5000 ng/mL, about0.01 and about 500 ng/mL, about 0.1 and about 100 ng/mL, or about 1 andabout 50 ng/mL.

In some embodiments, a cytokine is introduced into the cultured mediumat a concentration of at least 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 100, 200, 300, 400, 500, 1000 ng/mL or more. In someembodiments, a cytokine is introduced into the cultured medium at aconcentration of at most 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 100, 200, 300, 400, 500, 1000 ng/mL or less.

In some embodiments, OSM is introduced into the cultured medium at aconcentration of between about 0.001 and about 5000 ng/mL, about 0.01and about 500 ng/mL, about 0.1 and about 100 ng/mL, or about 1 and about50 ng/mL.

In some embodiments, OSM is introduced into the cultured medium at aconcentration of at least 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 100, 200, 300, 400, 500, 1000 ng/mL or more. In some embodiments,OSM is introduced into the cultured medium at a concentration of at most0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500,1000 ng/mL or less.

In some embodiments, a fibroblast growth factor (e.g. bFGF) modulatesthe expression of a biomarker within an hTS cell. In some embodiments,the biomarker is a microRNA (miR). In some embodiments, the biomarker ismiRNA-124a. In some embodiments, the fibroblast growth factor is bFGF.In some embodiments, bFGF upregulates or activates miRNA-124a. In someembodiments, bFGF downregulates miRNA-124a.

In some embodiments, the expression level of miRNA-124a in a bFGFtreated trophoblast stem cell is compared to the expression level ofmiRNA-124a in an untreated trophoblast stem cell. In some embodiments,the expression level of miRNA-124a is at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 1000 fold, or more in a bFGF treatedtrophoblast stem cell relative to the expression level of miRNA-124a inan untreated trophoblast stem cell. In some embodiments, the expressionlevel of miRNA-124a is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 100, 1000 fold, or less in a bFGF treated trophoblast stem cellrelative to the expression level of miRNA-124a in an untreatedtrophoblast stem cell.

In some embodiments, activation or upregulation of miRNA-124a initiatesdefinitive endoderm (DE) specification in the trophoblast stem cell. Insome embodiments, the DE specification occurs between about 0.1 andabout 96 hours, about 0.5 and about 36 hours, about 1 and about 24hours, about 2 and about 18 hours, about 4 and about 12 hours, or about6 and about 10 hours.

In some embodiments, the DE specification occurs at least 0.1, 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 36, 48, 72 hours, or more after induction with bFGF. In someembodiments, the DE specification occurs at most 0.1, 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 36, 48, 72 hours, or less after induction with bFGF. In someembodiments, the DE specification occurs about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours post induction with bFGF.In some embodiments, the DE specification occurs about 6, 7, 8, 9, or 10hours post induction with bFGF.

In some embodiments, the DE specification is associated with a set ofbiomarkers. In some embodiments, the biomarkers include forkhead boxprotein A2 (FOXA2), SRY-box 17 (SOX17), Goosecoid (GSC), Homeodomainprotein MIXL1, SRY-box 2 (SOX2), transcription factor NANOG, and OCT4.In some embodiments, the DE specification is characterized by anelevated expression of biomarkers selected from forkhead box protein A2(FOXA2), SRY-box 17 (SOX17), Goosecoid (GSC), Homeodomain protein MIXL1,SRY-box 2 (SOX2), transcription factor NANOG, and OCT4. In someembodiments, the DE specification is characterized by an elevatedexpression of biomarkers selected from forkhead box protein A2 (FOXA2),SRY-box 17 (SOX17), Goosecoid (GSC), SRY-box 2 (SOX2), transcriptionfactor NANOG, and OCT4. In some embodiments, the DE specification ischaracterized by a decreased expression of biomarkers selected fromforkhead box protein A2 (FOXA2), SRY-box 17 (SOX17), Goosecoid (GSC),Homeodomain protein MIXL1, SRY-box 2 (SOX2), transcription factor NANOG,and OCT4. In some embodiments, the DE specification is characterized bya decreased expression of Homeodomain protein MIXL1.

In some embodiments, the expression levels of FOXA2, SOX17, GSC, MIXL1,SOX2, NANOG, and OCT4 in a bFGF induced trophoblast stem cell arecompared to the expression levels of FOXA2, SOX17, GSC, MIXL1, SOX2,NANOG, and OCT4 in an uninduced trophoblast stem cell. In someembodiments, the elevated expression level is an increased proteinexpression level. In some embodiments, the protein expression levels ofFOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 are between about 1 andabout 20,000 fold, about 2 and about 1000 fold, or about 10 and about100 fold higher than the protein expression levels of FOXA2, SOX17, GSC,MIXL1, SOX2, NANOG, and OCT4 in an untreated trophoblast stem cell. Insome embodiments, the protein expression levels of FOXA2, SOX17, GSC,MIXL1, SOX2, NANOG, and OCT4 are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 100, 1000, 10,000 fold, or more in a bFGF treatedtrophoblast stem cell relative to the protein expression levels ofFOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 in an untreatedtrophoblast stem cell. In some embodiments, the protein expressionlevels of FOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 are at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 10,000 fold, orless in a bFGF treated trophoblast stem cell relative to the proteinexpression levels of FOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 inan untreated trophoblast stem cell.

In some embodiments, the elevated expression level is an increased geneexpression level. In some embodiments, the gene expression levels ofFOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 are between about 1 andabout 20,000 fold, about 2 and about 1000 fold, or about 10 and about100 fold higher than the gene expression levels of FOXA2, SOX17, GSC,MIXL1, SOX2, NANOG, and OCT4 in an untreated trophoblast stem cell. Insome embodiments, the gene expression levels of FOXA2, SOX17, GSC,MIXL1, SOX2, NANOG, and OCT4 are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 100, 1000, 10,000 fold, or more in a bFGF treatedtrophoblast stem cell relative to the gene expression levels of FOXA2,SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 in an untreated trophoblaststem cell. In some embodiments, the gene expression levels of FOXA2,SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 are at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 10,000 fold, or less in a bFGFtreated trophoblast stem cell relative to the gene expression levels ofFOXA2, SOX17, GSC, MIXL1, SOX2, NANOG, and OCT4 in an untreatedtrophoblast stem cell.

In some embodiments, a cocktail of a steroid and a cytokine is utilizedto direct DE differentiation into hepatic lineage. In some embodiments,the cocktail comprise dexamethasone and oncostatin M is utilized todirect DE differentiation into hepatic lineage. In some embodiments, thecocktail is introduced into a cultured medium comprising hTS cellsbefore, after, or simultaneously with the addition of bFGF. In someembodiments, the cocktail is introduced into a cultured mediumcomprising hTS cells between about 0.5 and about 96 hours, about 1 andabout 48 hours, about 2 and about 36 hours, about 3 and about 24 hours,about 4 and about 12 hours, or about 6 and about 10 hours.

In some embodiments, the cocktail is introduced into a cultured mediumcomprising hTS cells at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72hours, or more after addition of bFGF. In some embodiments, the cocktailis introduced into a cultured medium comprising hTS cells at most 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 36, 48, 60, 72 hours, or less after addition of bFGF. Insome embodiments, the cocktail is introduced into a cultured mediumcomprising hTS cells about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 hours after addition of bFGF. In some embodiments, thecocktail is introduced into a cultured medium comprising hTS cells about6, 7, 8, 9, 10, 11, or 12 hours after addition of bFGF.

In some embodiment, a hTS cell is incubated in a cultured mediumcomprising bFGF, dexamethasone and oncostatin M for between about 0.5and about 100 days, about 1 and about 50 days, about 2 and about 30days, about 3 and about 15 days, or about 4 and about 12 days.

In some embodiment, an hTS cell is incubated in a cultured mediumcomprising bFGF, dexamethasone and oncostatin M for at least 0.5 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In someembodiment, an hTS cell is incubated in a cultured medium comprisingbFGF, dexamethasone and oncostatin M for at most 0.5 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 days or less.

In some embodiments, the trophoblast stem cell is classified into fourstages of hepatocyte-like cell development. In some embodiments, thefour stages include primitive streak to DE stage, hepatic specifiedendoderm, hepatoblastic stage, and the fetal and adult hepatocyte-likecell stage. In some embodiments, each of the four stages are associatedwith a set of biomarkers comprising C—X—C chemokine receptor type 4(CXCR4), Forkhead box protein A2 (FOXA2), SRY-box 17 (SOX17),hexosaminidase A (alpha polypeptide) (HHEX), bile salt export pump(BSEP), transthyretin (TTR), albumin (ALB), tyrosine aminotransferase(TAT), cytochrome P450 7A1 (CYP7A1), glucose-6-phosphatase (G6PC),serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin)member 1 (SERPINA1), ATP-binding cassette sub-family C (ABCC2),CCAAT-enhancer-binding protein beta (C/EBPβ), hepatocyte nuclear factor1-alpha (HNF1α), hepatocyte nuclear factor 4-alpha (HNF4α),alpha-1-fetoprotein (AFP), keratin 8 (KRT8), phosphoenolpyruvatecarboxykinase 2 mitochondrial (PCK2), cytochrome P450 2B6 (CYP2B6),glycogen synthase 2 (GYS2), hepatocyte nuclear factor 6 (HNF6),carbamoyl-phosphate synthase 1 mitochondrial (CPS1), alcoholdehydrogenase 1C (class I) gamma polypeptide (ADH1C), connexin 32(CX32), cytochrome P450 3A4 (CYP3A4), prospero homeobox 1 (PROX1),tryptophan 2,3-dioxygenase (TDO2), apolipoprotein F (APOF), keratin 18(KRT18), keratin 19 (KRT19), or chromosome 19 open reading frame 80(angiopoietin-like protein 8, hepatocellular carcinoma-associated geneTD26, lipasin) (Betatrophin). In some embodiments, each of the fourstages are associated with a set of biomarkers comprising CXCR4, FOXA2,SOX17, HHEX, TTR, ALB, TAT, CYP7A1, BSEP, SERPINA1, G6PC, ABCC2, C/EBPβ,HNF1α, or HNF4α.

In some instances, the primitive streak to DE stage is a stage in whichan hTS cell that has not entered a hepatic differentiation stage or isabout to enter a hepatic differentiation stage. In some embodiments, anhTS cell at the primitive streak to DE stage undergoes DE specification.In some instances after induction with a FGF (e.g. bFGF), a hTS cellremains in the primitive streak to DE stage for between about 0.1 andabout 24 hours, about 0.5 and about 18 hours, or about 1 and about 12hours. In some instances after induction with a FGF (e.g. bFGF), a hTScell remains in the primitive streak to DE stage for at most 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or less. In some instances afterinduction with a FGF (e.g. bFGF), an hTS cell remains in the primitivestreak to DE stage for at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10hours or more.

In some instances, a hTS cell in the primitive streak to DE stage ischaracterized with a set of biomarkers selected from CXCR4, FOXA2,SOX17, HHEX, BSEP, TTR, ALB, TAT, CYP7A1, G6PC, SERPINA1, ABCC2, C/EBPβ,HNF1α, HNF4α, AFP, KRT8, PCK2, CYP2B6, GYS2, HNF6, CPS1, ADH1C, CX32,CYP3A4, PROX1, TDO2, APOF, KRT18, KRT19, and Betatrophin. In someembodiments, the primitive streak to DE stage is associated with a setof biomarkers selected from CXCR4, FOXA2, SOX17 and HHEX.

In some embodiments, the primitive streak to DE stage is associated withelevated expression levels of CXCR4, FOXA2, SOX17 and HHEX. In someembodiments, the elevated expression level is an increased proteinexpression level or an increased gene expression level. In someembodiments, the elevated expression level is an increased geneexpression level. In some embodiments, the elevated gene expressionlevels of CXCR4, FOXA2, SOX17 and HHEX are relative to the geneexpression levels of CXCR4, FOXA2, SOX17 and HHEX in an equivalenttrophoblast stem cell which has not entered the primitive streak to DEstage. In some embodiments, the elevated gene expression levels ofCXCR4, FOXA2, SOX17 and HHEX are between about 0.1 and about 10,000,about 1 and about 5000, or about 2 and about 1000. In some embodiments,the elevated gene expression levels of CXCR4, FOXA2, SOX17 and HHEX areat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000fold, or more relative to the gene expression levels of CXCR4, FOXA2,SOX17 and HHEX in an equivalent hTS cell which has not entered theprimitive streak to DE stage. In some embodiments, the gene expressionlevels of CXCR4, FOXA2, SOX17 and HHEX are at most 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000 fold, or less relative to thegene expression levels of CXCR4, FOXA2, SOX17 and HHEX in an equivalenthTS cell which has not entered the primitive streak to DE stage.

In some instances, the hepatic specified endoderm characterizes thefirst appearance of epithelium cells after hepatic specification. Insome embodiments, the hepatic specified endoderm stage is initiated withthe addition of a cocktail of steroid (e.g. dexamethasone) and cytokine(e.g. oncostatin M). In some instances, the cocktail of steroid (e.g.dexamethasone) and cytokine (e.g. oncostatin M) is added to the culturedmedium at between about 1 and about 48 hours, about 2 and about 24hours, about 3 and about 18 hours, or about 4 and about 12 hours. Insome instances, the cocktail of steroid (e.g. dexamethasone) andcytokine (e.g. oncostatin M) is added to the cultured medium at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 hours, or more postinduction of a FGF (e.g. bFGF). In some instances, the cocktail ofsteroid (e.g. dexamethasone) and cytokine (e.g. oncostatin M) is addedto the cultured medium at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 hours, or less post induction of a FGF (e.g. bFGF).

In some cases after induction with the cocktail of steroid (e.g.dexamethasone) and cytokine (e.g. oncostatin M), a hTS cell remains inthe hepatic specified endoderm stage for between about 1 and about 72hours, about 2 and about 36 hours, about 3 and about 24 hours, or about4 and about 12 hours. In some cases after induction with the cocktail ofsteroid (e.g. dexamethasone) and cytokine (e.g. oncostatin M), a hTScell remains in the hepatic specified endoderm stage for at least 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 hours, or more. In some casesafter induction with the cocktail of steroid (e.g. dexamethasone) andcytokine (e.g. oncostatin M), a hTS cell remains in the hepaticspecified endoderm stage for at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36 hours, or less.

In some instances, a hTS cell in the hepatic specified endoderm stage ischaracterized with a set of biomarkers selected from CXCR4, FOXA2,SOX17, HHEX, BSEP, TTR, ALB, TAT, CYP7A1, G6PC, SERPINA1, ABCC2, C/EBPβ,HNF1α, HNF4α, AFP, KRT8, PCK2, CYP2B6, GYS2, HNF6, CPS1, ADH1C, CX32,CYP3A4, PROX1, TDO2, APOF, KRT18, KRT19, and Betatrophin. In someembodiments, the hepatic specified endoderm is associated with a set ofbiomarkers selected from SOX17, TTR, ALB, TAT, and CYP7A1.

In some embodiments, the hepatic specified endoderm is associated withelevated expression levels of SOX17, TTR, ALB, TAT, and CYP7A1. In someembodiments, the elevated expression level is an increased proteinexpression level or an increased gene expression level. In someembodiments, the elevated expression level is an increased geneexpression level. In some embodiments, the elevated gene expressionlevels of SOX17, TTR, ALB, TAT, and CYP7A1 are relative to the geneexpression levels of SOX17, TTR, ALB, TAT, and CYP7A1 in an equivalenttrophoblast stem cell which has not entered the hepatic specifiedendoderm stage. In some embodiments, the gene expression levels ofSOX17, TTR, ALB, TAT, and CYP7A1 are between about 1 and about 10,000fold, about 2 and about 1000 fold, or about 2 and about 100 fold. Insome embodiments, the gene expression levels of SOX17, TTR, ALB, TAT,and CYP7A1 are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,100, 1000, 5000 fold, or more relative to the gene expression levels ofSOX17, TTR, ALB, TAT, and CYP7A1 in an equivalent trophoblast stem cellwhich has not entered the hepatic specified endoderm stage. In someembodiments, the gene expression levels of SOX17, TTR, ALB, TAT, andCYP7A1 are at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,1000, 5000 fold, or less relative to the gene expression levels ofSOX17, TTR, ALB, TAT, and CYP7A1 in an equivalent trophoblast stem cellwhich has not entered the hepatic specified endoderm stage.

In some embodiments, a hTS cell enters the hepatoblastic stage afterbetween about 1 and about 36 hours, about 3 and about 24 hours, or about6 and about 12 hours post addition of a cocktail of steroid (e.g.dexamethasone) and cytokine (e.g. oncostatin M). In some embodiments, ahTS cell enters the hepatoblastic stage after about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24hours post addition of a cocktail of steroid (e.g. dexamethasone) andcytokine (e.g. oncostatin M).

In some embodiments, an hTS cell remains in the hepatoblastic stage forbetween about 1 and about 30 days, about 2 and about 12 days, or about 3and about 7 days. In some embodiments, an hTS cell remains in thehepatoblastic stage for at most 1, 2, 3, 4, 5, 6, 7, 8 days or less. Insome embodiments, an hTS cell remains in the hepatoblastic stage for atleast 1, 2, 3, 4, 5, 6, 7, 8 days or more.

In some instances, a hTS cell in the hepatoblastic stage ischaracterized with a set of biomarkers selected from CXCR4, FOXA2,SOX17, HHEX, BSEP, TTR, ALB, TAT, CYP7A1, G6PC, SERPINA1, ABCC2, C/EBPβ,HNF1α, HNF4α, AFP, KRT8, PCK2, CYP2B6, GYS2, HNF6, CPS1, ADH1C, CX32,CYP3A4, PROX1, TDO2, APOF, KRT18, KRT19, and Betatrophin. In someembodiments, the hepatoblastic stage is associated with a set ofbiomarkers selected from TTR, ALB, TAT, CYP7A1, and BSEP.

In some embodiments, the hepatoblastic stage is associated with elevatedexpression levels of TTR, ALB, TAT, CYP7A1, and BSEP. In someembodiments, the elevated expression level is an increased proteinexpression level or an increased gene expression level. In someembodiments, the elevated expression level is an increased geneexpression level. In some embodiments, the elevated gene expressionlevels of TTR, ALB, TAT, CYP7A1, and BSEP are relative to the geneexpression levels of TTR, ALB, TAT, CYP7A1, and BSEP in an equivalenthTS cell which has not entered the hepatoblastic stage. In someembodiments, the gene expression levels of TTR, ALB, TAT, CYP7A1, andBSEP are between about 1 and about 10,000 fold, about 2 and about 1000fold, or about 2 and about 100 fold.

In some embodiments, the gene expression levels of TTR, ALB, TAT,CYP7A1, and BSEP are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 100, 200, 300, 400, 500, 1000, 5000 fold, or more relative to thegene expression levels of TTR, ALB, TAT, CYP7A1, and BSEP in anequivalent hTS cell which has not entered the hepatoblastic stage. Insome embodiments, the gene expression levels of TTR, ALB, TAT, CYP7A1,and BSEP are at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100,200, 300, 400, 500, 1000, 5000 fold, or less relative to the geneexpression levels of TTR, ALB, TAT, CYP7A1, and BSEP in an equivalenthTS cell which has not entered the hepatoblastic stage.

In some instances, a hTS cell enters the fetal and adult hepatocyte-likestage between about 1 and about 20 days, about 2 and about 10 days, orabout 3 and about 6 days post addition of a cocktail of steroid (e.g.dexamethasone) and cytokine (e.g. oncostatin M). In some instances, anhTS cell enters the fetal and adult hepatocyte-like stage after about 1,2, 3, 4, 5, or 6 days after post addition of a cocktail of steroid (e.g.dexamethasone) and cytokine (e.g. oncostatin M).

In some embodiments, an hTS cell remains in the fetal and adulthepatocyte-like stage for between about 1 and about 100 days, about 2and about 50 days, about 3 and about 30 days, about 4 and about 12 days,or about 6 and about 10 days. In some embodiments, an hTS cell remainsin the fetal and adult hepatocyte-like stage for at most 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 60 days or less. In someembodiments, an hTS cell remains in the fetal and adult hepatocyte-likestage for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20, 25, 30, 60 days or more.

In some instances, a hTS cell in the fetal and adult hepatocyte-likestage is characterized with a set of biomarkers selected from CXCR4,FOXA2, SOX17, HHEX, BSEP, TTR, ALB, TAT, CYP7A1, G6PC, SERPINA1, ABCC2,C/EBPβ, HNF1α, HNF4α, AFP, KRT8, PCK2, CYP2B6, GYS2, HNF6, CPS1, ADH1C,CX32, CYP3A4, PROX1, TDO2, APOF, KRT18, KRT19, and Betatrophin. In someembodiments, the fetal and adult hepatocyte-like cell stage isassociated with a set of biomarkers selected from HHEX, BSEP, TTR, ALB,TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α.

In some embodiments, the fetal and adult hepatocyte-like cell stage isassociated with elevated expression levels of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α. In some embodiments,the elevated expression level is an increased protein expression levelor an increased gene expression level. In some embodiments, the elevatedexpression level is an increased gene expression level. In someembodiments, the elevated gene expression levels of HHEX, BSEP, TTR,ALB, TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α are relativeto the gene expression level of HHEX, BSEP, TTR, ALB, TAT, SERPINA1,G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α in an equivalent hTS cell whichhas not entered the fetal and adult hepatocyte-like cell stage. In someembodiments, the gene expression levels of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α are between about 1 andabout 10,000 fold, about 2 and about 5000 fold, or about 2 and about1000 fold.

In some embodiments, the gene expression levels of HHEX, BSEP, TTR, ALB,TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α are at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000 fold, or morerelative to the gene expression levels of HHEX, BSEP, TTR, ALB, TAT,SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α in an equivalent hTScell which has not entered the fetal and adult hepatocyte-like cellstage. In some embodiments, the gene expression levels of HHEX, BSEP,TTR, ALB, TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α are atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1000, 5000fold, or less relative to the gene expression levels of HHEX, BSEP, TTR,ALB, TAT, SERPINA1, G6PC, ABCC2, C/EBPβ, HNF1α, and HNF4α in anequivalent hTS cell which has not entered the fetal and adulthepatocyte-like cell stage.

In some embodiments, a cell in the fetal and adult hepatocyte-like cellstage matures into an induced hepatocyte. In some embodiments, a cell inthe fetal and adult hepatocyte-like cell stage is an induced hepatocyte.In some embodiments, the induced hepatocyte does not differentiatefurther. In some embodiments, the induced hepatocyte reaches aterminally differentiated stage. In some embodiments, the inducedhepatocyte remains as a stable cell for up to 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100generations or more. In some embodiments, the induced hepatocyte remainsas a stable cell for up to 7 days, 10 days, 14 days, 21 days, 30 days,60 days, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 1 years, 2 years, or more.

Methods of Use

Described herein, in certain embodiments, are methods of utilizinginduced hepatocytes for one or more uses. In some embodiments, inducedhepatocytes are utilized for screening a compound for use in treatmentor prevention of a disease or disorder. In some embodiments, thescreening process involves one or more of studies of the compound,enzyme induction, and toxicity studies. In some embodiments, inducedhepatocytes are administered for the treatment of a liver injury, suchas a damage or loss of liver tissue due to a liver-associated disease ordisorder. In other embodiments, induced hepatocytes are utilized for theproduction of therapeutic proteins (e.g. hormones), cytokines,cholesterols, carbohydrates, bile, or a combination thereof. In otherembodiments, induced hepatocytes are utilized for liver regeneration. Inadditional embodiments, induced hepatocytes are utilized as a source forgene therapy.

Induced Hepatocytes for Screening a Compound

In some embodiments, induced hepatocytes are utilized for screening acompound for use in treatment or prevention of a disease or disorder. Insome embodiments, the method comprises contacting an isolated inducedhepatocyte with a compound. In some embodiments, the method comprisescontacting an isolated primary hepatocyte with the compound. In otherembodiments, the method further comprises detecting a change in theactivity of at least one biomarker (e.g. gene, transcript or protein) inthe induced hepatocytes. In other embodiments, the method furthercomprises detecting a change in the level of at least one biomarker(e.g. gene, transcript or protein) in primary hepatocytes. In someembodiments, a change in the level of at least one biomarker is a changein the gene expression level of at least one biomarker. In someembodiments, the biomarker comprises a member of the cytochrome P450superfamily. In some embodiments, the biomarker comprises cytochromeP450 families: CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17,CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46, CYP51, and theirrespective subfamily members. In some embodiments, the biomarkercomprises CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, orCYP7A1.

In some embodiments, a compound (e.g. a drug) is metabolized through theliver. In some instances, the liver's primary mechanism for metabolizingthe compound (e.g. drug) is the P450 cytochrome system of enzymes. Insome instances, the rate of metabolizing the compound is too quickly, itmay decrease the compound's efficacy. Alternatively, if the rate ofmetabolizing the compound is too slow, it may allow toxicity to build upin the host cell. Therefore in some instances, the metabolism of acompound is evaluated, such as for example, its toxicity.

In some instances, the compound acts as an inducer or an inhibitortoward a member of the cytochrome P450 family of enzymes. In some cases,the compound is an inducer toward a member of the cytochrome P450 familyof enzymes. In some instances, an inducer initiates or enhances theexpression level of an enzyme, such as a member of the cytochrome P450family of enzymes. In some instances, an inducer initiates or enhancesthe gene expression level of a member of the cytochrome P450 family ofenzymes. In some instances, an inducer initiates or enhances the proteinexpression level of a member of the cytochrome P450 family of enzymes.In some cases, the compound is an inhibitor toward a member of thecytochrome P450 family of enzymes. In some instances, an inhibitorinhibits, decreases, or interferes with the expression level of anenzyme, such as a member of the cytochrome P450 family of enzymes. Insome instances, an inhibitor inhibits, decreases, or interferes with thegene expression level of a member of the cytochrome P450 family ofenzymes. In some instances, an inhibitor inhibits, decreases, orinterferes with the protein expression level of a member of thecytochrome P450 family of enzymes. In some instances, the expressionlevel of the enzyme, such as a member of the cytochrome P450 family ofenzymes, is compared to a control expression level of the enzyme. Insome embodiments, the control expression level of the enzyme is theexpression level of the enzyme uninduced by the compound.

In some instances after contacting an isolated induced hepatocyte with acompound, a change in the gene expression of a member of the cytochromeP450 superfamily is detected. In some cases, the gene expression levelof a biomarker from a member of the cytochrome P450 superfamily from anisolated induced hepatocyte that have been contacted with a compound iscompared to the gene expression level of a biomarker from the cytochromeP450 superfamily of an equivalent isolated induced hepatocyte notcontacted with the compound or compared with a primary hepatocyte.

In some instances after contacting an isolated induced hepatocyte with acompound, a change in the gene expression level of a biomarker selectedfrom cytochrome P450 families: CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8,CYP11, CYP17, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46,CYP51, and their respective subfamily members is detected. In somecases, the gene expression level of a biomarker from the cytochrome P450families: CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19,CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46, CYP51, and theirrespective subfamily members from an isolated induced hepatocyte thathave been contacted with a compound is compared to the gene expressionlevel of a biomarker from the cytochrome P450 families: CYP1, CYP2,CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19, CYP20, CYP21, CYP24,CYP26, CYP27, CYP39, CYP46, CYP51, and their respective subfamilymembers of an equivalent isolated induced hepatocyte not contacted withthe compound or compared with a primary hepatocyte.

In some instances after contacting an isolated induced hepatocyte with acompound, a change in the gene expression level of a biomarker selectedfrom CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP7A1is detected. In some cases, the gene expression level of a biomarkerselected from CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4,and CYP7A1 from an isolated induced hepatocyte that have been contactedwith a compound is compared to the gene expression level of a biomarkerselected from CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4,and CYP7A1 of an equivalent isolated induced hepatocyte not contactedwith the compound or compared with a primary hepatocyte.

In one embodiment, provided herein describes a method of screening acompound for the ability to induce changes in a cell. In one embodiment,the method comprises contacting an isolated induced hepatocyte with thecompound. In another embodiment, the method comprises contacting anisolated primary hepatocyte with the compound. In another embodiment,the method further comprises detecting an induction of change (e.g.proliferation) of the induced hepatocyte such as during regeneration. Inanother embodiment, the method further comprises detecting an inductionof change (e.g. proliferation) of the primary hepatocyte such as duringregeneration.

Also provided herein a method of screening a compound for cellulartoxicity or modulation of the cell, the method comprising contacting aninduced hepatocyte with the compound. In another embodiment, the methodfurther comprises determining any phenotypic or metabolic changes in thecell that result from contact with the compound, and correlating thechange with cellular toxicity or any other change in cell function orbiochemistry. In another embodiment, screening of pharmaceuticals,toxins, or potential modulators of differentiation is facilitated. Thesesubstances (e.g., pharmaceuticals, toxins, or potential modulators) canbe added to the culture medium.

One embodiment provided herein described a method of screeningproliferation factors, differentiation factors, and pharmaceuticals. Inone embodiment, induced hepatocytes or primary hepatocytes are used toscreen for factors (such as small molecule drugs, peptides,polynucleotides, and the like) or conditions (such as culture conditionsor manipulation) that affect the characteristics of induced hepatocytesor primary hepatocytes in culture. In one embodiment, this system hasthe advantage of not being complicated by a secondary effect caused byperturbation of the feeder cells by the test compound. In anotherembodiment, growth affecting substances are tested. In anotherembodiment, the conditioned medium is withdrawn from the culture and asimpler medium is substituted. In another embodiment, different wellsare then treated with different cocktails of soluble factors that arecandidates for replacing the components of the conditioned medium.Efficacy of each mixture is determined if the treated cells aremaintained and proliferate in a satisfactory manner, optimally as wellas in conditioned medium. Potential differentiation factors orconditions can be tested by treating the cell according to the testprotocol, and then determining whether the treated cell developsfunctional or phenotypic characteristics of a differentiated cell of aparticular lineage.

In one embodiment, the induced hepatocyte or primary hepatocyte are usedto screen potential modulators of cellular differentiation. For example,in one assay for screening modulators of cellular differentiation, theinduced hepatocyte or primary hepatocyte can be cultured under serumfree, or in the present of a modulator, as the situation requires, andthe effect on differentiation can be detected. In another embodiment,the screening methods described herein can be used to study conditionsassociated with cellular development and screen for potentialtherapeutic or corrective drugs or modulators of the condition. Forexample, in one embodiment, the development of the induced hepatocyte orprimary hepatocyte is compared with the development with cells having adisease or condition.

In one embodiment, biomarker such as gene and protein expression can becompared between different cell populations obtained from inducedhepatocyte or primary hepatocyte, and used to identify and characterizefactors upregulated or downregulated in the course of proliferation, andproduce nucleotide copies of the affected genes.

In one embodiment, feeder-free induced hepatocyte or primary hepatocytecultures can also be used for the testing of pharmaceutical compounds indrug research. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the induced hepatocyte or primaryhepatocyte with the candidate compound, determining any resultingchange, and then correlating the effect of the compound with theobserved change. In another embodiment, the screening is done, forexample, either because the compound is designed to have apharmacological effect on certain cell types, or because a compounddesigned to have effects elsewhere have unintended side effects. Inanother embodiment, two or more drugs are be tested in combination (bycombining with the cells either simultaneously or sequentially), todetect possible drug-drug interaction effects. In another embodiment,compounds are screened initially for potential toxicity. In anotherembodiment, cytotoxicity is be determined by the effect on cellviability, survival, morphology, on the expression or release of certainmarkers, receptors or enzymes, on DNA synthesis or repair.

The terms “treating,” “treatment,” and the like are used herein to meanobtaining a desired pharmacologic and/or physiologic effect. In someembodiments, an individual (e.g., an individual suspected to besuffering from and/or genetically pre-disposed to a liver-associateddisease or disorder is treated prophylactically with a preparation ofinduced hepatocyte described herein and such prophylactic treatmentcompletely or partially prevents a liver-associated disease or disorderor sign or symptom thereof. In some embodiments, an individual istreated therapeutically (e.g., when an individual is suffering from aliver-associated disease or disorder), such therapeutic treatment causesa partial or complete cure for the disease or disorder and/or reversesan adverse effect attributable to the disease or disorder and/orstabilizes the disease or disorder and/or delays progression of thedisease or disorder and/or causes regression of the disease or disorder.

Administration (e.g., transplantation) of induced hepatocyte to the areain need of treatment is achieved by, for example and not by way oflimitation, local infusion during surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

“Transplanting” a composition into a mammal refers to introducing thecomposition into the body of the mammal by any method established in theart. The composition being introduced is the “transplant”, and themammal is the “recipient”. The transplant and the recipient can besyngeneic, allogeneic or xenogeneic. Further, the transplantation can bean autologous transplantation.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. For example, an effective amount ofinduced hepatocytes is an amount sufficient, as the case can be, toresult in an increase in primary hepatocyte number. An effective amountof a composition to treat or ameliorate a liver-associated disease ordisorder is an amount of the composition sufficient to reduce or removethe symptoms of the liver-associated disease or disorder. The effectiveamount of a given therapeutic agent will vary with factors such as thenature of the agent, the route of administration, the size and speciesof the animal to receive the therapeutic agent, and the purpose of theadministration.

Further provided herein in one embodiment are genetically modifiedinduced hepatocytes. Manipulations modify various properties of thecell, e.g., render it more adapted or resistant to certain environmentalconditions, and/or induce a production of one or more certain substancestherefrom, which substances can, e.g., improve the viability of thecell. Such genetic alterations can be performed in order to make thecell more suitable for use in transplantation, for example, in order toavoid rejection thereof from the recipient (for reviews of gene therapyprocedures, see Anderson, Science, 256:808; Mulligan, Science, 926;Miller, Nature, 357:455; Van Brunt, Biotechnology, 6(10):1149; and Yu etal., Gene Therapy, 1:13).

A “vector” refers to a recombinant DNA or RNA construct, such as aplasmid, a phage, recombinant virus, or other vector that, uponintroduction into an appropriate host cell, results in a modification ofa progenitor cell described herein. Appropriate expression vectors arewell known to those with ordinary skill in the art and include thosethat are replicable in eukaryotic and/or prokaryotic cells and thosethat remain episomal or those that integrate into the host cell genome.

Construction of vectors is achieved using techniques described in, forexample, as described in Sambrook et al., 1989. In one embodimentisolated plasmids or DNA fragments are cleaved, tailored, and relegatedin the form desired to generate the plasmids. If desired, analysis toconfirm correct sequences in the constructed plasmids is performed usingany suitable method. Suitable methods for constructing expressionvectors, preparing in vitro transcripts, introducing DNA into hostcells, and performing analyses for assessing gene expression andfunction are known. Gene presence, amplification, and/or expression aremeasured in a sample directly, for example, by conventional Southernblotting, Northern blotting to quantitate the transcription of mRNA, dotblotting (DNA or RNA analysis), or in situ hybridization, using anappropriately labeled probe which can be based on a sequence providedherein.

As used herein, terms such as “transfection”, “transformation”, and thelike are intended to indicate the transfer of nucleic acid to a cell ororganism in functional form. Such terms include various means oftransferring nucleic acids to cells, including transfection with CaPO4,electroporation, viral transduction, lipofection, delivery usingliposomes, and/or other delivery vehicles.

Induced Hepatocytes for Treatment of a Disease or Disorder

In some embodiments, induced hepatocytes are administered for thetreatment of a liver injury. In some embodiments, liver injury includesdamaged or loss of liver tissue due to external factors, such as injuryto the host, e.g. injury to an individual, or due to surgery. In someembodiments, liver injury includes damage or loss of liver tissue due toa liver-associated disease or disorder. In some embodiments, theliver-associated disease or disorder is an acute liver disease ordisorder such as acute liver failure, or is a chronic liver disease ordisorder such as cirrhosis. In some embodiments, the liver-associateddisease or disorder is resulted from genetic factors, chemicals orpathogenic infections.

Exemplary liver-associated diseases or disorders include, but are notlimited to, alagille syndrome, alpha 1 anti-trypsin deficiency,autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis,cystic disease of the liver, fatty liver disease includingalcohol-related liver disease and non-alcohol fatty liver disease(NAFLD), galactosemia, gallstones, Gilbert's Syndrome, hemochromatosis,liver cysts, liver cancer, liver disease in pregnancy (e.g. acute fattyliver of pregnancy, intrahepatic cholestasis of pregnancy, preeclampsia,or HELLP Syndrome (hemolysis, elevated liver tests, low platelets)),neonatal hepatitis, primary billary cirrhosis, primary sclerosingcholangitis, porphyria, Reye's Syndrome, sarcoidosis, toxic hepatitis,type 1 glocogen storage disease, tyrosinemia, viral hepatitis A, B, C,and Wilson disease.

In some embodiments, induced hepatocytes are administered for thetreatment of an acute liver disease or disorder. In other embodiments,induced hepatocytes are administered for the treatment of a chronicliver disease or disorder. In some embodiments, induced hepatocytes areadministered for the treatment of alagille syndrome, alpha 1anti-trypsin deficiency, autoimmune hepatitis, benign liver tumors,biliary atresia, cirrhosis, cystic disease of the liver, fatty liverdisease including alcohol-related liver disease and non-alcohol fattyliver disease (NAFLD), galactosemia, gallstones, Gilbert's Syndrome,hemochromatosis, liver cysts, liver cancer, liver disease in pregnancy(e.g. acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, preeclampsia, or HELLP Syndrome (hemolysis, elevated livertests, low platelets)), neonatal hepatitis, primary billary cirrhosis,primary sclerosing cholangitis, porphyria, Reye's Syndrome, sarcoidosis,toxic hepatitis, type 1 glocogen storage disease, tyrosinemia, viralhepatitis A, B, C, Wilson disease, or a combination thereof.

In some embodiments, induced hepatocytes are administered in combinationwith an additional therapeutic agent for the treatment of aliver-associated disease or disorder. In some embodiments, theadditional therapeutic agent includes, but is not limited to, curcumin,resveratrol, thalidomide, cholestyramine (Questran), tacrolimus(PROGRAF), ursodiol (Actigall), interferons, diuretics such as loopdiuretics, and liver transplantation.

In some embodiments, chemicals that are toxic to the liver results inchemical-induced liver disease. In some embodiments, chemicals that aretoxic to the liver include drugs; consumables such as alcohols, foodadditives, or preservatives; and chemical and environmental toxins. Insome instances, drugs that cause liver injury include, but are notlimited to, acetaminophen, allopurinol, anabolic steroids, danazol,dantrolene, imipramine, isoniazid, methotrexate, methyldopa, nicotinicacid, nitrofurantoin, phenothiazines, phenytoin, salicylates, statins,terbinafine HCl, thiabendazole, thorotrast, tolbutamide,chlorpromazine/valproic acid, and chlorpropamide/erythro-mycin.

In some embodiments, pathogenic infections induce liver-associateddiseases or disorders. In some embodiments, a pathogen is a virus, abacterium, a fungus, or a parasite. In some embodiments, the pathogen isa virus. In some embodiments, a viral infection induces liver-associateddiseases or disorders.

In some embodiments, a virus is a DNA virus or an RNA virus. In someinstances, a DNA virus is a single-stranded (ss) DNA virus, adouble-stranded (ds) DNA virus, or a DNA virus that contains both ss andds DNA regions. In some cases, an RNA virus is a single-stranded (ss)RNA virus or a double-stranded (ds) RNA virus. Sometimes, a ssRNA virusis further classified into a positive-sense RNA virus or anegative-sense RNA virus.

In some instances, a dsDNA virus is from the family: Myoviridae,Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae,Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae,Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae,Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae,Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae,Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae,Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae,Poxviridae, Sphaerolipoviridae, and Tectiviridae.

In some instances, an ssDNA virus is from the family: Anelloviridae,Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae,Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.

In some instances, a DNA virus that contains both ss and ds DNA regionsis from the group of pleolipoviruses. In some cases, the pleolipovirusesinclude Haloarcula hispanica pleomorphic virus 1, Halogeometricumpleomorphic virus 1, Halorubrum pleomorphic virus 1, Halorubrumpleomorphic virus 2, Halorubrum pleomorphic virus 3, and Halorubrumpleomorphic virus 6.

In some instances, a dsRNA virus is from the family: Birnaviridae,Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae,Megavirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae,Rotavirus and Totiviridae.

In some cases, a positive-sense ssRNA virus is from the family:Alphaflexiviridae, Alphatetraviridae, Alvernaviridae, Arteriviridae,Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae,Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae,Dicistroviridae, Flaviviridae, Gammaflexiviridae, Iflaviridae,Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae,Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae,Roniviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae, andVirgaviridae.

Sometimes, a negative-sense ssRNA virus is from the family:Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae,Arenaviridae, Bunyaviridae, Ophioviridae, and Orthomyxoviridae.

Exemplary virus includes, but is not limited to: Abelson leukemia virus,Abelson murine leukemia virus, Abelson's virus, Acutelaryngotracheobronchitis virus, Adelaide River virus, Adeno associatedvirus group, Adenovirus, African horse sickness virus, African swinefever virus, AIDS virus, Aleutian mink disease parvovirus,Alpharetrovirus, Alphavirus, ALV related virus, Amapari virus,Aphthovirus, Aquareovirus, Arbovirus, Arbovirus C, arbovirus group A,arbovirus group B, Arenavirus group, Argentine hemorrhagic fever virus,Argentine hemorrhagic fever virus, Arterivirus, Astrovirus, Atelineherpesvirus group, Aujezky's disease virus, Aura virus, Ausduk diseasevirus, Australian bat lyssavirus, Aviadenovirus, avian erythroblastosisvirus, avian infectious bronchitis virus, avian leukemia virus, avianleukosis virus, avian lymphomatosis virus, avian myeloblastosis virus,avian paramyxovirus, avian pneumoencephalitis virus, avianreticuloendotheliosis virus, avian sarcoma virus, avian type Cretrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B19 virus,Babanki virus, baboon herpesvirus, baculovirus, Barmah Forest virus,Bebaru virus, Berrimah virus, Betaretrovirus, Birnavirus, Bittner virus,BK virus, Black Creek Canal virus, bluetongue virus, Bolivianhemorrhagic fever virus, Boma disease virus, border disease of sheepvirus, borna virus, bovine alphaherpesvirus 1, bovine alphaherpesvirus2, bovine coronavirus, bovine ephemeral fever virus, bovineimmunodeficiency virus, bovine leukemia virus, bovine leukosis virus,bovine mammillitis virus, bovine papillomavirus, bovine papularstomatitis virus, bovine parvovirus, bovine syncytial virus, bovine typeC oncovirus, bovine viral diarrhea virus, Buggy Creek virus, bulletshaped virus group, Bunyamwera virus supergroup, Bunyavirus, Burkitt'slymphoma virus, Bwamba Fever, CA virus, Calicivirus, Californiaencephalitis virus, camelpox virus, canarypox virus, canid herpesvirus,canine coronavirus, canine distemper virus, canine herpesvirus, canineminute virus, canine parvovirus, Cano Delgadito virus, caprine arthritisvirus, caprine encephalitis virus, Caprine Herpes Virus, Capripox virus,Cardiovirus, caviid herpesvirus 1, Cercopithecid herpesvirus 1,cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2, Chandipuravirus, Changuinola virus, channel catfish virus, Charleville virus,chickenpox virus, Chikungunya virus, chimpanzee herpesvirus, chubreovirus, chum salmon virus, Cocal virus, Coho salmon reovirus, coitalexanthema virus, Colorado tick fever virus, Coltivirus, Columbia SKvirus, common cold virus, contagious eethyma virus, contagious pustulardermatitis virus, Coronavirus, Corriparta virus, coryza virus, cowpoxvirus, coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricketparalysis virus, Crimean-Congo hemorrhagic fever virus, croup associatedvirus, Cryptovirus, Cypovirus, Cytomegalovirus, cytomegalovirus group,cytoplasmic polyhedrosis virus, deer papillomavirus, deltaretrovirus,dengue virus, Densovirus, Dependovirus, Dhori virus, diploma virus,Drosophila C virus, duck hepatitis B virus, duck hepatitis virus 1, duckhepatitis virus 2, duovirus, Duvenhage virus, Deformed wing virus DWV,eastern equine encephalitis virus, eastern equine encephalomyelitisvirus, EB virus, Ebola virus, Ebola-like virus, echo virus, echovirus,echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus,EIA virus, EIA virus, encephalitis virus, encephalomyocarditis groupvirus, encephalomyocarditis virus, Enterovirus, enzyme elevating virus,enzyme elevating virus (LDH), epidemic hemorrhagic fever virus,epizootic hemorrhagic disease virus, Epstein-Barr virus, equidalphaherpesvirus 1, equid alphaherpesvirus 4, equid herpesvirus 2,equine abortion virus, equine arteritis virus, equine encephalosisvirus, equine infectious anemia virus, equine morbillivirus, equinerhinopneumonitis virus, equine rhinovirus, Eubenangu virus, European elkpapillomavirus, European swine fever virus, Everglades virus, Eyachvirus, felid herpesvirus 1, feline calicivirus, feline fibrosarcomavirus, feline herpesvirus, feline immunodeficiency virus, felineinfectious peritonitis virus, feline leukemia/sarcoma virus, felineleukemia virus, feline panleukopenia virus, feline parvovirus, felinesarcoma virus, feline syncytial virus, Filovirus, Flanders virus,Flavivirus, foot and mouth disease virus, Fort Morgan virus, FourCorners hantavirus, fowl adenovirus 1, fowlpox virus, Friend virus,Gammaretrovirus, GB hepatitis virus, GB virus, German measles virus,Getah virus, gibbon ape leukemia virus, glandular fever virus, goatpoxvirus, golden shinner virus, Gonometa virus, goose parvovirus,granulosis virus, Gross' virus, ground squirrel hepatitis B virus, groupA arbovirus, Guanarito virus, guinea pig cytomegalovirus, guinea pigtype C virus, Hantaan virus, Hantavirus, hard clam reovirus, harefibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2,hemagglutinating virus of Japan, hemorrhagic fever virus, hendra virus,Henipaviruses, Hepadnavirus, hepatitis A virus, hepatitis B virus group,hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis Evirus, hepatitis F virus, hepatitis G virus, hepatitis nonA nonB virus,hepatitis virus, hepatitis virus (nonhuman), hepatoencephalomyelitisreovirus 3, Hepatovirus, heron hepatitis B virus, herpes B virus, herpessimplex virus, herpes simplex virus 1, herpes simplex virus 2,herpesvirus, herpesvirus 7, Herpesvirus ateles, Herpesvirus hominis,Herpesvirus infection, Herpesvirus saimiri, Herpesvirus suis,Herpesvirus varicellae, Highlands J virus, Hirame rhabdovirus, hogcholera virus, human adenovirus 2, human alphaherpesvirus 1, humanalphaherpesvirus 2, human alphaherpesvirus 3, human B lymphotropicvirus, human betaherpesvirus 5, human coronavirus, human cytomegalovirusgroup, human foamy virus, human gammaherpesvirus 4, humangammaherpesvirus 6, human hepatitis A virus, human herpesvirus 1 group,human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus4 group, human herpesvirus 6, human herpesvirus 8, humanimmunodeficiency virus, human immunodeficiency virus 1, humanimmunodeficiency virus 2, human papillomavirus, human T cell leukemiavirus, human T cell leukemia virus I, human T cell leukemia virus II,human T cell leukemia virus III, human T cell lymphoma virus I, human Tcell lymphoma virus II, human T cell lymphotropic virus type 1, human Tcell lymphotropic virus type 2, human T lymphotropic virus I, human Tlymphotropic virus II, human T lymphotropic virus III, Ichnovirus,infantile gastroenteritis virus, infectious bovine rhinotracheitisvirus, infectious haematopoietic necrosis virus, infectious pancreaticnecrosis virus, influenza virus A, influenza virus B, influenza virus C,influenza virus D, influenza virus pr8, insect iridescent virus, insectvirus, iridovirus, Japanese B virus, Japanese encephalitis virus, JCvirus, Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovovirus, Kilham's rat virus, Klamath virus, Kolongo virus, Koreanhemorrhagic fever virus, kumba virus, Kysanur forest disease virus,Kyzylagach virus, La Crosse virus, lactic dehydrogenase elevating virus,lactic dehydrogenase virus, Lagos bat virus, Langur virus, lapineparvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus,Leaky virus, Lentivirus, Leporipoxvirus, leukemia virus, leukovirus,lumpy skin disease virus, lymphadenopathy associated virus,Lymphocryptovirus, lymphocytic choriomeningitis virus,lymphoproliferative virus group, Machupo virus, mad itch virus,mammalian type B oncovirus group, mammalian type B retroviruses,mammalian type C retrovirus group, mammalian type D retroviruses,mammary tumor virus, Mapuera virus, Marburg virus, Marburg-like virus,Mason Pfizer monkey virus, Mastadenovirus, Mayaro virus, ME virus,measles virus, Menangle virus, Mengo virus, Mengovirus, Middelburgvirus, milkers nodule virus, mink enteritis virus, minute virus of mice,MLV related virus, MM virus, Mokola virus, Molluscipoxvirus, Molluscumcontagiosum virus, monkey B virus, monkeypox virus, Mononegavirales,Morbillivirus, Mount Elgon bat virus, mouse cytomegalovirus, mouseencephalomyelitis virus, mouse hepatitis virus, mouse K virus, mouseleukemia virus, mouse mammary tumor virus, mouse minute virus, mousepneumonia virus, mouse poliomyelitis virus, mouse polyomavirus, mousesarcoma virus, mousepox virus, Mozambique virus, Mucambo virus, mucosaldisease virus, mumps virus, murid betaherpesvirus 1, muridcytomegalovirus 2, murine cytomegalovirus group, murineencephalomyelitis virus, murine hepatitis virus, murine leukemia virus,murine nodule inducing virus, murine polyomavirus, murine sarcoma virus,Muromegalovirus, Murray Valley encephalitis virus, myxoma virus,Myxovirus, Myxovirus multiforme, Myxovirus parotitidis, Nairobi sheepdisease virus, Nairovirus, Nanirnavirus, Nariva virus, Ndumo virus,Neethling virus, Nelson Bay virus, neurotropic virus, New WorldArenavirus, newborn pneumonitis virus, Newcastle disease virus, Nipahvirus, noncytopathogenic virus, Norwalk virus, nuclear polyhedrosisvirus (NPV), nipple neck virus, O'nyong'nyong virus, Ockelbo virus,oncogenic virus, oncogenic viruslike particle, oncornavirus, Orbivirus,Orf virus, Oropouche virus, Orthohepadnavirus, Orthomyxovirus,Orthopoxvirus, Orthoreovirus, Orungo, ovine papillomavirus, ovinecatarrhal fever virus, owl monkey herpesvirus, Palyam virus,Papillomavirus, Papillomavirus sylvilagi, Papovavirus, parainfluenzavirus, parainfluenza virus type 1, parainfluenza virus type 2,parainfluenza virus type 3, parainfluenza virus type 4, Paramyxovirus,Parapoxvirus, paravaccinia virus, Parvovirus, Parvovirus B19, parvovirusgroup, Pestivirus, Phlebovirus, phocine distemper virus, Picodnavirus,Picornavirus, pig cytomegalovirus-pigeonpox virus, Piry virus, Pixunavirus, pneumonia virus of mice, Pneumovirus, poliomyelitis virus,poliovirus, Polydnavirus, polyhedral virus, polyoma virus, Polyomavirus,Polyomavirus bovis, Polyomavirus cercopitheci, Polyomavirus hominis 2,Polyomavirus maccacae 1, Polyomavirus muris 1, Polyomavirus muris 2,Polyomavirus papionis 1, Polyomavirus papionis 2, Polyomavirussylvilagi, Pongine herpesvirus 1, porcine epidemic diarrhea virus,porcine hemagglutinating encephalomyelitis virus, porcine parvovirus,porcine transmissible gastroenteritis virus, porcine type C virus, poxvirus, poxvirus, poxvirus variolae, Prospect Hill virus, Provirus,pseudocowpox virus, pseudorabies virus, psittacinepox virus, quailpoxvirus, rabbit fibroma virus, rabbit kidney vaculolating virus, rabbitpapillomavirus, rabies virus, raccoon parvovirus, raccoonpox virus,Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus,Rauscher's virus, recombinant vaccinia virus, recombinant virus,reovirus, reovirus 1, reovirus 2, reovirus 3, reptilian type C virus,respiratory infection virus, respiratory syncytial virus, respiratoryvirus, reticuloendotheliosis virus, Rhabdovirus, Rhabdovirus carpia,Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus,Riley's virus, rinderpest virus, RNA tumor virus, Ross River virus,Rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubeolavirus, Rubivirus, Russian autumn encephalitis virus, SA 11 simian virus,SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1,salivary gland virus, sandfly fever virus group, Sandjimba virus, SARSvirus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki ForestVirus, Seoul virus, sheeppox virus, Shope fibroma virus, Shope papillomavirus, simian foamy virus, simian hepatitis A virus, simian humanimmunodeficiency virus, simian immunodeficiency virus, simianparainfluenza virus, simian T cell lymphotrophic virus, simian virus,simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpoxvirus, South American hemorrhagic fever viruses, sparrowpox virus,Spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV 1virus group, STLV (simian T lymphotropic virus) type I, STLV (simian Tlymphotropic virus) type II, STLV (simian T lymphotropic virus) typeIII, stomatitis papulosa virus, submaxillary virus, suidalphaherpesvirus 1, suid herpesvirus 2, Suipoxvirus, swamp fever virus,swinepox virus, Swiss mouse leukemia virus, TAC virus, Tacaribe complexvirus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus,Theiler's encephalomyelitis virus, Theiler's virus, Thogoto virus,Thottapalayam virus, Tick borne encephalitis virus, Tioman virus,Togavirus, Torovirus, tumor virus, Tupaia virus, turkey rhinotracheitisvirus, turkeypox virus, type C retroviruses, type D oncovirus, type Dretrovirus group, ulcerative disease rhabdovirus, Una virus, Uukuniemivirus group, vaccinia virus, vacuolating virus, varicella zoster virus,Varicellovirus, Varicola virus, variola major virus, variola virus,Vasin Gishu disease virus, VEE virus, Venezuelan equine encephalitisvirus, Venezuelan equine encephalomyelitis virus, Venezuelan hemorrhagicfever virus, vesicular stomatitis virus, Vesiculovirus, Vilyuisk virus,viper retrovirus, viral haemorrhagic septicemia virus, Visna Maedivirus, Visna virus, volepox virus, VSV (vesicular stomatitis virus),Wallal virus, Warrego virus, wart virus, WEE virus, West Nile virus,western equine encephalitis virus, western equine encephalomyelitisvirus, Whataroa virus, Winter Vomiting Virus, woodchuck hepatitis Bvirus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yabamonkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus, andthe Yug Bogdanovac virus.

In some embodiments, a viral infection induces a liver-associateddisease or disorder. In some embodiments, the viral infection is ahepatitis infection. In some embodiments, the virus is a hepatitis Avirus, a hepatitis B virus, a hepatitis C virus, a hepatitis D virus, ahepatitis E virus, a hepatitis F virus, or a hepatitis G virus. In someembodiments, a viral infection that induces a liver-associated diseaseor disorder is caused by a hepatitis A virus, a hepatitis B virus, ahepatitis C virus, a hepatitis D virus, a hepatitis E virus, a hepatitisF virus, or a hepatitis G virus.

In some embodiments, liver injury arises as a consequence of an immuneresponse to virus within the liver. In some instances, liver injuryarises as part of a generalized host infection in which viruses targettissues other than the liver. Exemplary viruses those primary target isnot liver include herpes viruses such as Epstein-Barr virus,cytomegalovirus (CMV) or herpes simplex virus; parvovirus; adenovirus;influenza viruses; lentivirus such as human immunodeficiency virus(HIV); and severe acute respiratory syndrome (SARS)-associatedcoronavirus. In some instances, viral infections described hereinresults in hepatitis.

In some embodiments, the pathogen is a bacterium. In some embodiments, abacterial infection induces liver-associated diseases or disorders.Examples of bacteria include: Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M.avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracis, Corynebacterium diphtherias, Corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidum, Treponema pertenue, Leptospira, andActinomyces israelli.

In some embodiments, the pathogen is a fungus. In some embodiments, afungal infection induces liver-associated diseases or disorders.Examples of fungi include: Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (i.e.,protists) include: Plasmodium falciparum and Toxoplasma gondii.

In some embodiments, the pathogen is a parasite. In some embodiments, aparasitic infection induces liver-associated diseases or disorders.Examples of parasite include: sarcodina (e.g. Entamoeba), mastigophora(e.g. Giardia, Leishmania), ciliophora (e.g. Balantidium), and sporozoa(e.g. Plasmodium, Cryptosporidium).

In some embodiments, induced hepatocytes are utilized for regenerationof a defect liver. In some instances, induced hepatocytes are introducedat the site of liver injury. In some cases, induced hepatocytes areintroduced or transplanted as a cell suspension to the site of liverinjury. In other cases, induced hepatocytes are introduced ortransplanted as a tissue mass to the site of liver injury. In otherinstances, induced hepatocytes are utilized for regeneration of a defectliver ex vivo. In other instances, induced hepatocytes are utilized forex vivo liver generation prior to transplantation into a site of liverinjury.

In some embodiments, induced hepatocytes are formulated as acomposition, such as a pharmaceutical composition, for treating a liverinjury due to external factors, such as injury to the host, e.g. injuryto an individual, or due to surgery.

In some embodiments, induced hepatocytes are formulated as acomposition, such as a pharmaceutical composition, for treating a liverinjury due to a liver-associated disease or disorder. In someembodiments, induced hepatocytes are formulated as a composition, suchas a pharmaceutical composition, for treating alagille syndrome, alpha 1anti-trypsin deficiency, autoimmune hepatitis, benign liver tumors,biliary atresia, cirrhosis, cystic disease of the liver, fatty liverdisease including alcohol-related liver disease and non-alcohol fattyliver disease (NAFLD), galactosemia, gallstones, Gilbert's Syndrome,hemochromatosis, liver cysts, liver cancer, liver disease in pregnancy(e.g. acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, preeclampsia, or HELLP Syndrome (hemolysis, elevated livertests, low platelets)), neonatal hepatitis, primary billary cirrhosis,primary sclerosing cholangitis, porphyria, Reye's Syndrome, sarcoidosis,toxic hepatitis, type 1 glocogen storage disease, tyrosinemia, viralhepatitis A, B, C, Wilson disease, or a combination thereof.

In some embodiments, the composition comprising induced hepatocytesfurther comprises an additional therapeutic agent. In some embodiment,the additional therapeutic agent is a therapeutic agent for thetreatment of a liver-associated disease or disorder. In someembodiments, the additional therapeutic agent includes, but is notlimited to, curcumin, resveratrol, thalidomide, cholestyramine(Questran), tacrolimus (PROGRAF), ursodiol (Actigall), interferons,diuretics such as loop diuretics, and liver transplantation.

In some embodiments, the composition comprising induced hepatocytes areutilized at the site of liver injury for regeneration of the liver. Insome embodiments, the composition comprising induced hepatocytes areintroduced or transplanted as a cell suspension to the site of liverinjury. In some embodiments, the composition comprising inducedhepatocytes are introduced or transplanted as a tissue mass to the siteof liver injury. In other embodiments, the composition comprisinginduced hepatocytes are utilized for regeneration of a defect liver exvivo. In other embodiments, the composition comprising inducedhepatocytes are utilized for ex vivo liver generation prior totransplantation into a site of liver injury.

Modes of administration of an isolated induced hepatocyte include, butare not limited to, systemic intravenous injection and injectiondirectly to the intended site of activity (e.g., endoscopic retrogradeinjection). The preparation can be administered by any convenient route,for example, by infusion or bolus injection, and can be administeredtogether with other biologically active agents. In some embodiments, theadministration is systemic localized administration.

In some embodiments, a composition comprising induced hepatocytes isformulated as a pharmaceutical composition for intravenousadministration to a mammal, including a human. In some embodiments,compositions for intravenous administration are solutions in steriletonic aqueous buffer. Where necessary, the composition also includes alocal anesthetic to ameliorate any pain at the site of the injection.Where the composition is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients are mixed prior to administration.

In some embodiments, suitable pharmaceutical compositions comprise atherapeutically effective amount of the induced hepatocytes and apharmaceutically acceptable carrier or excipient. Such a carrierincludes, but is not limited to, saline, buffered saline, dextrose,water, and combinations thereof.

In some embodiments, the induced hepatocytes described herein aredelivered to a targeted site (e.g., a defect section of the liver) by adelivery system suitable for targeting cells to a particular tissue. Forexample, the cells are encapsulated in a delivery vehicle that allowsfor the slow release of the cell(s) at the targeted site. The deliveryvehicle is modified such that it is specifically targeted to aparticular tissue. The surface of the targeted delivery system ismodified in a variety of ways. In the case of a liposomal-targeteddelivery system, lipid groups are incorporated into the lipid bilayer ofthe liposome in order to maintain the targeting ligand in stableassociation with the liposomal bilayer.

In other examples, a colloidal dispersion system is used. Colloidaldispersion systems include macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems, including oil-in-wateremulsions, micelles, mixed micelles, and liposomes.

The administration of induced hepatocytes described herein is optionallytailored to an individual, by: (1) increasing or decreasing the amountcells injected; (2) varying the number of injections; or (3) varying themethod of delivery of the cells.

The induced hepatocyte preparation is used in an amount effective topromote engraftment of cells in the recipient. At the physician'sdiscretion, the administration is adjusted to meet optimal efficacy andpharmacological dosing.

Induced Hepatocytes for Production of Therapeutic Proteins

In some embodiments, induced hepatocytes are utilized for the productionof therapeutic proteins (e.g. hormones), cytokines, cholesterols,carbohydrates, bile, or a combination thereof. In some instances,induced hepatocytes are utilized for the production of therapeuticproteins. In some instances, the therapeutic proteins include fulllength proteins, domains or fragments thereof, or peptides. In someinstances, the proteins, domains or fragments thereof, or peptidescontaining natural and/or unnatural amino acid residues. In some cases,the therapeutic proteins, their fragments thereof, or peptides, include,but are not limited to, major plasma proteins such as human serumalbumin, soluble plasma fibronectin, α-fetoprotein, C-reactive protein,and several globulins; proteins involved in hemostasis and fibrinolysissuch as coagulation factors involved in the coagulation cascade,α2-macroglobulin, α1-antitrypsin, antithrombin III, protein S, proteinC, plasminogen, α2-antiplasmin, and complement component 3; carrierproteins such as albumin, ceruloplasmin, transcortin, haptoglobin,hemopexin, IGF binding rotein, major urinary proteins, retinol bindingprotein, sex hormone-binding globulin, transthyretin, transferrin, andVitamin D-binding protein; hormones such as insulin-like growth factor1, thrombopoietin, hepcidin, and betatrophin; prohormones such asangiotensinogen; and apolipoproteins. In some embodiments, inducedhepatocytes are utilized for the production of hormones or its fragmentsthereof. In some embodiments, induced hepatocytes are utilized for theproduction of insulin-like growth factor 1, thrombopoietin, hepcidin,betatrophin, angiotensinogen, or their fragments thereof.

In some instances, induced hepatocytes are utilized for the productionof cytokines. As described elsewhere herein, cytokines includechemokines, interferons, interleukins, and tumor necrosis factors. Insome embodiments, cytokines are proinflammatory cytokines. In someembodiments, the cytokines include C—X—C and C—C subfamilies ofchemokines: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7,CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17,CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27,CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9,CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17. Insome embodiments, the cytokines include growth related (GRO)-alpha,GRO-beta, GRO-gamma, epithelial neutrophile activating peptide-78(ENA-78), RANTES, TNF-alpha, IL-1β, IL-6, IL-8, MCP-1, M-CSF, IFN-α,IFN-β, and cytokine-induced neutrophil chemoattractant (CINC). In someinstances, induced hepatocytes are utilized for the production of one ormore cytokines described herein. In some embodiments, inducedhepatocytes are utilized for the production of cytokines including, butnot limiting to: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6,CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16,CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26,CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17,growth related (GRO)-alpha, GRO-beta, GRO-gamma, epithelial neutrophileactivating peptide-78 (ENA-78), RANTES, TNF-alpha, IL-1β, IL-6, IL-8,MCP-1, M-CSF, IFN-α, IFN-β, and cytokine-induced neutrophilchemoattractant (CINC).

In some instances, induced hepatocytes are utilized for the productionor process of cholesterol. As used herein, the term “cholesterol”includes cholesterol, its stereoisomers (e.g. nat-cholesterol, orent-cholesterol), naturally occurring cholesterols, genetically modifiedcholesterol, or their fragments thereof. In some embodiments, inducedhepatocytes are utilized for the production or process ofnat-cholesterol or its fragments thereof. In some embodiments, inducedhepatocytes are utilized for the production or process ofent-cholesterol or its fragments thereof.

In some instances, induced hepatocytes are utilized for the productionor process of carbohydrates. In some instances, induced hepatocytes areutilized for the formation, breakdown or interconversion ofcarbohydrates, gluconeogenesis, glycogenolysis, glycogenesis, lipidmetabolism including cholesterol synthesis as disclosed above, andlipogenesis.

In some instances, induced hepatocytes are utilized for the productionof bile.

As used herein, an amino acid residue can refer to a molecule containingboth an amino group and a carboxyl group. Suitable amino acids include,without limitation, both the D- and L-isomers of the naturally-occurringamino acids, as well as non-naturally occurring amino acids prepared byother metabolic routes. The term amino acid, as used herein, includes,without limitation, α-amino acids, natural amino acids, non-naturalamino acids, and amino acid analogs.

The term “α-amino acid” can refer to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon.

The term “β-amino acid” can refer to a molecule containing both an aminogroup and a carboxyl group in a β configuration.

“Naturally occurring amino acid” can refer to any one of the twentyamino acids commonly found in peptides synthesized in nature, and knownby the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M,F, P, S, T, W, Y and V.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptidomimetic macrocycle Amino acid analogsinclude, without limitation, β-amino acids and amino acids where theamino or carboxy group is substituted by a similarly reactive group(e.g., substitution of the primary amine with a secondary or tertiaryamine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is notone of the twenty amino acids commonly found in peptides synthesized innature, and known by the one letter abbreviations A, R, N, C, D, Q, E,G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acidsinclude, without limitation, p-acetylphenylalanine,m-acetylphenylalanine, p-(3-oxbutanoyl)-1-phenylalanine,p-(2-amino-3-hydroxyethyl)phenylalanine,p-isopropylthiocarbonyl-phenylalanine,p-ethylthiocarbonyl-phenylalanine, o-propargyloxyphenylalanine,p-azidophehylalanine, phenylselenidylalanine, p-benzoyl-1-phenylalanine,or p-boronophenylalanine.

Induced Hepatocytes for Liver Regeneration

In other embodiments, induced hepatocytes are utilized for liverregeneration. In other embodiments, induced hepatocytes are utilized forex-vivo liver regeneration. In some cases, the ex-vivo liverregeneration utilizes an ex vivo perfusion system. In some instances,the ex vivo perfusion system include ex vivo perfusion bioreactor system(e.g. a 3D perfusion bioreactor system). In some cases, the ex-vivoliver regeneration utilizes a bioprinting system. In some instances, thebioprinting system is a 3 dimensional (3D) bioprinting system. In somecases, the ex-vivo liver regeneration utilizes a 3D bioprinting system.In some embodiments, a 3D bioprinting system includes Organovo's NovoGEnMMX Bioprinter, EnvisionTEC's BioPlotter®, GeSims BioScaffolder 2.1, orregenHu's BioFactory®. In some embodiments, a 3D bioprinting systemincludes the 3D printing technology from OxSyBio.

In some embodiments, a 3D bioprinting system includes the systemdescribed in: U.S. Pat. No. 8,691,974, U.S. Pat. No. 8,691,274,US20140052285, US20140012407, US20140099709, US20140093932,US20130304233, US20130004469, US20130017564, US20130164339,US20120089238, US20110250688, US20090208466, EP2679669, WO2014039427,WO2013181375, WO2013040087, and WO2012122105.

In some embodiments, induced hepatocytes are utilized for liverregeneration using a perfusion system (e.g. an ex vivo perfusionsystem). In some embodiments, induced hepatocytes are utilized for liverregeneration using an ex vivo perfusion bioreactor system (e.g. a 3Dperfusion bioreactor system). In some instances, induced hepatocytes areutilized for liver regeneration using a bioprinting system (e.g. a 3Dbioprinting system). In some instances, induced hepatocytes are utilizedfor liver regeneration using Organovo's NovoGEn MMX Bioprinter,EnvisionTEC's BioPlotter®, GeSims BioScaffolder 2.1, or regenHu'sBioFactory®. In some instances, induced hepatocytes are utilized forliver regeneration using the 3D printing technology from OxSyBio.

In some cases, induced hepatocytes are utilized for liver regenerationusing a 3D bioprinting system described in: U.S. Pat. No. 8,691,974,U.S. Pat. No. 8,691,274, US20140052285, US20140012407, US20140099709,US20140093932, US20130304233, US20130004469, US20130017564,US20130164339, US20120089238, US20110250688, US20090208466, EP2679669,WO2014039427, WO2013181375, WO2013040087, or WO2012122105.

In some instances, induced hepatocytes are utilized for generation oftissue scaffolds through one or more of the methods described above. Insome instances, the tissue scaffold is a 2 dimensional (2D) tissuescaffold or a 3 dimensional (3D) tissue scaffold. In some instances, thetissue scaffold is a 3D tissue scaffold. In some instances, inducedhepatocytes are utilized for generation of 3D tissue scaffolds. In someembodiments, the 3D-scaffold is pre-coated with one or moreextracellular matrix components, e.g., gelatin, laminin, fibronectin,collagen, polylysine, vitronectin, hyaluronic acid, hyaluronanhydrogels, silk fibroin, chitosan or a composite of any of theforementioned. In some embodiments, the cells are seeded at higherdensity on the 3D scaffolds than on the 2D cultures, such as threefoldhigher or fivefold higher or tenfold higher. In some embodiments, thecells are seeded in the presence of cell survival factor, such as aninhibitor of ROCK Rho kinase.

In some aspects, a biocompatible substrate is used to generate thetissue herein, e.g., culturing cells under a culture medium on a threedimensional biocompatible substrate. In some embodiments, thebiocompatible substrate comprises a polymeric substrate. In someembodiments, the biocompatible substrate is biodegradable. In someembodiments, the polymeric substrate comprises poly(caprolactone) (PCL).In some embodiments, the polymeric substrate is selected from the groupconsisting of polylactic acid (PLA), poly-L-lactic acid (PLLA),poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA),polylactide-co-glycolide (PLGA), polydioxanone, polygluconate,polylactic acid-polyethylene oxide copolymers, modified cellulose,collagen, polyhydroxybutyrate, polyhydroxpriopionic acid,polyphosphoester, poly(alpha-hydroxy acid), polycarbonates, polyamides,polyanhydrides, polyamino acids, polyorthoesters, polyacetals,polycyanoacrylates, degradable urethanes, aliphatic polyesterpolyacrylates, polymethacrylate, acyl substituted cellulose acetates,non-degradable polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins,polyethylene oxide, polyvinyl alcohol, Teflon®, nylon silicon,poly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys and oligo(ε-caprolactone)diol. In some embodiments, thebiocompatible substrate comprises a synthetic polymer. According to someembodiments of the invention, the biocompatible substrate comprises anatural polymer. In some embodiments, the biodegradable substrate isselected from the group consisting of poly(caprolactone) (PCL),polyglycolic acid, poly(DL-lactic-coglycolic acid), cat gut sutures,cotton, cellulose, gelatin, dextran, alginate, fibronectin, laminin,collagen, hyaluronic acid, polyhydroxyalkanoate, poly 4 hydroxybutirate(P4HB) and polygluconic acid (PGA).

Induced Hepatocytes as a Source for Gene Therapy

In additional embodiments, induced hepatocytes are utilized as a sourcefor gene therapy. In some instances, the gene therapy is an ex vivo genetherapy. In some instances, induced hepatocytes are utilized as atherapeutic agent for gene therapy treatment of a liver-associateddisease or disorder. In some instances, the gene therapy treatment is aninduced hepatocyte-based gene therapy. In some instances, the inducedhepatocyte-based gene therapy is utilized such as for example replacingdefective or missing gene products, preventing allograft rejection,repopulating host liver, aid in generating xenogeneic hepatocytes, ortailored for patient-specific liver treatment or regeneration.

As described elsewhere herein, a liver-associated disease or disorderincludes alagille syndrome, alpha 1 anti-trypsin deficiency, autoimmunehepatitis, benign liver tumors, biliary atresia, cirrhosis, cysticdisease of the liver, fatty liver disease including alcohol-relatedliver disease and non-alcohol fatty liver disease (NAFLD), galactosemia,gallstones, Gilbert's Syndrome, hemochromatosis, liver cysts, livercancer, liver disease in pregnancy (e.g. acute fatty liver of pregnancy,intrahepatic cholestasis of pregnancy, preeclampsia, or HELLP Syndrome(hemolysis, elevated liver tests, low platelets)), neonatal hepatitis,primary billary cirrhosis, primary sclerosing cholangitis, porphyria,Reye's Syndrome, sarcoidosis, toxic hepatitis, type 1 glocogen storagedisease, tyrosinemia, viral hepatitis A, B, C, and Wilson disease.

In some instances, induced hepatocytes are utilized as a therapeuticagent for gene therapy treatment of alagille syndrome, alpha 1anti-trypsin deficiency, autoimmune hepatitis, benign liver tumors,biliary atresia, cirrhosis, cystic disease of the liver, fatty liverdisease including alcohol-related liver disease and non-alcohol fattyliver disease (NAFLD), galactosemia, gallstones, Gilbert's Syndrome,hemochromatosis, liver cysts, liver cancer, liver disease in pregnancy(e.g. acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, preeclampsia, or HELLP Syndrome (hemolysis, elevated livertests, low platelets)), neonatal hepatitis, primary billary cirrhosis,primary sclerosing cholangitis, porphyria, Reye's Syndrome, sarcoidosis,toxic hepatitis, type 1 glocogen storage disease, tyrosinemia, viralhepatitis A, B, C, Wilson disease, or a combination thereof.

In some instances, genes are transferred into induced hepatocytes viaviral or nonviral means. In some instances, viral means include vectorssuch as from murine leukemia retroviruses and lentiviruses,adenoassociated virus, T-antigen-deleted SV40 virus, neurotropic virusessuch as Herpes simplex virus, episomal viruses and hybrid viruses suchas adenoassociated viruses. In some instances, nonviral means includelipoplexes, polyplexes, dendrimers, inorganic nanoparticles, or hybridvectors such as virosomes, which are a combinatino of liposomes with aninactivated virus such as HIV or influenza virus. In some instances,additional nonviral means include injection of naked DNA,electroporation, gene gun, sonoporation, or magnetofection. Methods ofgene transfer by viral or nonviral means are well known in the art andare described for example, in Guha et al. “Hepatocyte-based genetherapy,” J Hepatobillary Pancreat Surg. 8(1):51-57 (2001).

As used herein, a gene can contain at least two nucleotides linkedtogether. In some instances, a nucleic acid described herein can containphosphodiester bonds, natural nucleic acids, or unnatural nucleic acids.A natural nucleic acid include both deoxyribonucleic acid (DNA) androbonucleic acid (RNA) and is known by the one letter abbreviations A,T, G, C, and U. Exemplary unnatural nucleic acids include, withoutlimitation, diaminopurine, isoguanine, isocytosine,2-amino-6-(2-thienyl)purine, pyrrole-2-carbaldehyde,2,6-bis(ethylthiomethyl)pyridine, pyridine-2,6-dicarboxamide,4-methylbenzimidazole, 2,3-difluorotoluene, d5SICS, and dNaM.

Diagnostic Methods

Methods for determining the expression or presence of biomarkersdescribed supra are well known in the art, and can be measured, forexample, by flow cytometry, immunohistochemistry, Western Blot,immunoprecipitation, magnetic bead selection, and quantification ofcells expressing either of these cell surface markers. Biomarker RNAexpression levels could be measured by RT-PCR, Qt-PCR, microarray,Northern blot, or other similar technologies.

By “detecting expression” or detecting “expression levels” is intendedfor determining the expression level or presence of a biomarker proteinor gene in the biological sample. Thus, “detecting expression”encompasses instances where a biomarker is determined not to beexpressed, not to be detectably expressed, expressed at a low level,expressed at a normal level, or overexpressed.

In some embodiments, the expression or presence of a biomarker describedherein is determined at a nucleic acid level, using, for example,immunohistochemistry techniques or nucleic acid-based techniques such asin situ hybridization and RT-PCR. In one embodiments, the expression orpresence of one or more biomarkers is carried out by a means for nucleicacid amplification, a means for nucleic acid sequencing, a meansutilizing a nucleic acid microarray (DNA and RNA), or a means for insitu hybridization using specifically labeled probes.

In other embodiments, the determining the expression or presence of abiomarker is carried out through gel electrophoresis. In one embodiment,the determination is carried out through transfer to a membrane andhybridization with a specific probe.

In other embodiments, the determining the expression or presence of abiomarker is carried out by a diagnostic imaging technique.

In still other embodiments, the determining the expression or presenceof a biomarker is carried out by a detectable solid substrate. In oneembodiment, the detectable solid substrate is paramagnetic nanoparticlesfunctionalized with antibodies.

In some embodiments, the expression or presence of a biomarker is at anRNA (e.g. mRNA) level. In some embodiments, techniques that detect RNA(e.g. mRNA) level include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays.

One method for the detection of mRNA levels involves contacting theisolated mRNA with a nucleic acid molecule (probe) that hybridize to themRNA encoded by the gene being detected. The nucleic acid probecomprises of, for example, a full-length cDNA, or a portion thereof,such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to an mRNA or genomic DNA encoding a biomarkerdescribed herein. Hybridization of an mRNA with the probe indicates thatthe biomarker or other target protein of interest is being expressed.

In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan readilyadapts known mRNA detection methods for use in detecting the level ofmRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of an mRNA of interestin a sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189 193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In some embodiments, biomarker expression is assessed byquantitative fluorogenic RT-PCR (i.e., the TaqMan0 System).

Expression levels of an RNA of interest are monitored using a membraneblot (such as used in hybridization analysis such as Northern, dot, andthe like), or microwells, sample tubes, gels, beads or fibers (or anysolid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The detection of expression alsocomprises using nucleic acid probes in solution.

In some embodiments, microarrays are used to determine expression orpresence of one or more biomarkers. Nucleic acid microarrays provide onemethod for the simultaneous measurement of the expression levels oflarge numbers of genes. Each array consists of a reproducible pattern ofcapture probes attached to a solid support. Labeled RNA or DNA ishybridized to complementary probes on the array and then detected bylaser scanning Hybridization intensities for each probe on the array aredetermined and converted to a quantitative value representing relativegene expression levels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and6,020,135, 6,033,860, and 6,344,316, which are incorporated herein byreference. High-density oligonucleotide arrays are particularly usefulfor determining the gene expression profile for a large number of RNA'sin a sample.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261, incorporatedherein by reference in its entirety. In some embodiments, an array isfabricated on a surface of virtually any shape or even a multiplicity ofsurfaces. In some embodiments, an array is a planar array surface. Insome embodiments, arrays include peptides or nucleic acids on beads,gels, polymeric surfaces, fibers such as fiber optics, glass or anyother appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporatedin its entirety for all purposes. In some embodiments, arrays arepackaged in such a manner as to allow for diagnostics or othermanipulation of an all-inclusive device.

In some embodiments, the expression or presence of a biomarker describedherein is determined at a protein level, using, for example, antibodiesthat are directed against specific biomarker proteins. These antibodiesare used in various methods such as Western blot, ELISA, multiplexingtechnologies, immunoprecipitation, or immunohistochemistry techniques.In some embodiments, detection of biomarkers is accomplished by ELISA.In some embodiments, detection of biomarkers is accomplished byelectrochemiluminescence (ECL).

Any means for specifically identifying and quantifying a biomarker inthe biological sample is contemplated. Thus, in some embodiments,expression level of a biomarker protein of interest in a biologicalsample is detected by means of a binding protein capable of interactingspecifically with that biomarker protein or a biologically activevariant thereof. In some embodiments, labeled antibodies, bindingportions thereof, or other binding partners are used. The word “label”when used herein refers to a detectable compound or composition that isconjugated directly or indirectly to the antibody so as to generate a“labeled” antibody. In some embodiments, the label is detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, catalyzes chemical alteration of a substratecompound or composition that is detectable.

The antibodies for detection of a biomarker protein are eithermonoclonal or polyclonal in origin, or are synthetically orrecombinantly produced. The amount of complexed protein, for example,the amount of biomarker protein associated with the binding protein, forexample, an antibody that specifically binds to the biomarker protein,is determined using standard protein detection methodologies known tothose of skill in the art. A detailed review of immunological assaydesign, theory and protocols are found in numerous texts in the art(see, for example, Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY));Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley& Sons, Inc., New York, N.Y.).

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of the marker is readilydeterminable to one skilled in the art. These labeled antibodies areused in immunoassays as well as in histological applications to detectthe presence of any biomarker or protein of interest. The labeledantibodies are either polyclonal or monoclonal. Further, the antibodiesfor use in detecting a protein of interest are labeled with aradioactive atom, an enzyme, a chromophoric or fluorescent moiety, or acolorimetric tag as described elsewhere herein. The choice of tagginglabel also will depend on the detection limitations desired. Enzymeassays (ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate.Radionuclides that serve as detectable labels include, for example,1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109. Examples of enzymes that serve as detectable labels include, butare not limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoricmoieties include, but are not limited to, fluorescein and rhodamine. Theantibodies are conjugated to these labels by methods known in the art.For example, enzymes and chromophoric molecules are conjugated to theantibodies by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Alternatively, conjugationoccurs through a ligand-receptor pair. Examples of suitableligand-receptor pairs are biotin-avidin or biotin-streptavidin, andantibody-antigen.

In certain embodiments, expression or presence of one or more biomarkersor other proteins of interest within a biological sample is determinedby radioimmunoassays or enzyme-linked immunoassays (ELISAs), competitivebinding enzyme-linked immunoassays, dot blot (see, for example, PromegaProtocols and Applications Guide, Promega Corporation (1991), Westernblot (see, for example, Sambrook et al. (1989) Molecular Cloning, ALaboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), chromatography such as high performance liquidchromatography (HPLC), or other assays known in the art. Thus, thedetection assays involve steps such as, but not limited to,immunoblotting, immunodiffusion, immunoelectrophoresis, orimmunoprecipitation.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods and compositions describedherein. Such kits include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of use.

For example, the container(s) include hTS cells, optionally in acomposition as disclosed herein. Such kits optionally include anidentifying description or label or instructions relating to its use inthe methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In some embodiments, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Cell Culture and Differentiation

Undifferentiated hTS cells were maintained in α-MEM (Gibco) supplementedwith 10% (v/v) fetal bovine serum (SAFC Biosciences). Cultures weremanually passaged at a 1:3-1:6 split ratio every 2-3 days.Differentiation of DE was carried out by a conditioned α-MEM mediacontaining 10% FBS, 1 mM 2-mercaptoethanol, 10 mM nicotinamide, and 10ng/ml bFGF for 8 hr in 37° C., 5% CO₂ incubator. For hepatocyticdifferentiation, cultured medium was added with dexamethasone (0.1 μM,Sigma) and recombinant human oncostatin M (10 ng/ml, Excel-BiomedicalInc.) and incubated for an additional 4 days or 6 days.

The study was approved by the Institutional Review Board on HumanSubjects Research and Ethics Committees, Kaohsiung Medical UniversityHospital. The hTS cells were obtained with informed consent.

Plasmids

MiR-124a precursor and anti-miR-124a were purchased from SystemBiosciences. Briefly, miR-124a precursor (60 pmol) or anti-miR-124a (60pmol) was transfected to hTS cells in 12-well culture dishes usingTransIT-LT1 transfection reagent (Minis, Madison, Wis.). Total RNAs wereused for quantifying miR-124a at 36 hr after transfection. Smallinterfering RNA (siRNA) targeting PI3K (SASI_Hs01_00233971 andSASI_Hs01_00127787), AKT1 (SASI_Hs01_00205545), and AKT2(SASI_Hs01_00035055) were purchased from Sigma. Short hairpin RNA(shRNA) targeting CREB1 (TRCN0000007310, TRCN0000226467 andTRCN0000226468), SMAD4 (TRCN0000010321, TRCN0000010323 andTRCN0000040032), AKT3 (TRCN0000001615 and TRCN0000001616), OCT4(TRCN0000004879 and TRCN0000004882), CDX2 (TRCN0000013683 andTRCN0000013686), and control shRNA (shGFP; TRCN0000072178,TRCN0000072179 and TRCN0000072183) were purchased from National RNAiCore platform, Academia Sinica, Taiwan. Transfection was performed withsiRNA or shRNA at 2 μg plus 4 μl transfection reagent.

Western Blot and Immunopreciptation (IP)

For immunoblotting assay, cells were harvested into RIPA lysis solution(Millipore, Billerica, Mass.) supplemented with protease and phosphataseinhibitors (Roche). After electrophoresis of 30 μg lysates onpolyacrylamide gels, electroblotting onto PDVF membranes (Millipore) wasperformed. After blocked by 5% non-fat milk in PBS at room temperature(1 hr), target protein was detected by using primary antibody. Allmembranes were incubated with chemiluminescent (Millipore) and imagingwas captured by the ChemiDoc XRS system (Bio-RAD). Antibodies used werelisted in Table 1. Data were analyzed by AlphaEaseFC (version 4.0.0)system. For IP assay, Cell lysates of bFGF-treated hTS cells werecollected. By incubation with protein G-agarose (Millipore) for 30 min,total protein (100 μg) was treated with specific primary antibodyovernight listed in Table 1. After treating with protein G-agarose beads(2 hr), sample was washed three times with RIPA lysis buffer(Millipore), following by adding with protein loading dye and boiled for5 min. The sample was resolved by 8% SDS-PAGE and subjected toimmunoblotting analysis.

mRNA, miRNA, and Chromatin Immunoprecipitation (CHiP)-qPCR Assays

For mRNA expression, RNA was isolated from hTS cells in triplicate orquintuple samples using TRIZOL reagent (Invitrogen) with DNAase Ion-column digestion (Qiagen, Valencia, Calif.). Total RNA (500 ng) wasused for reverse transcription with iScript cDNA synthesis kit(Bio-Rad). Real-time polymerase chain reaction (qPCR) was carried out induplicate using 1/40^(th) of the cDNA per reaction and 400 nM forwardand reverse primers. Comparative real-time PCR was carried out at leasttriplicate using the Power SYBR® Master Mix (Applied BioSystems) withthe 7500 Real-Time PCR System (Applied Biosystems). All genes werenormalized to the GAPDH expression and were normalized to the expressionof undifferentiated hTS cells using the ΔΔCt method. Primer sequencesused in this study are illustrated in Table 2.

For miRNA analysis, 25 ng of total RNA was reverse-transcribed using theTaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qPCR wascarried out at least triplicate using the TaqMan Universal PCR MasterMix (Applied Biosystems) with the 7500 Real-Time PCR System (AppliedBiosystems) including no-template controls, using specific primers formiR-124a or RNU6B (Applied Biosystems). U6 snRNA (RNU6B; AppliedBiosystems) served as an endogenous control.

For ChIP assay, hTS cell samples of indicated time in induction werefixed with a final concentration of 1% formaldehyde. After incubation atroom temperature (10 min), the reaction was stopped by adding glycine(125 mM). ChIP assay was performed using a protocol associated with theChIP assay kit (Upstate Biotechnology). After extensive washing, ChIPedDNA was eluted from the beads and analyzed by qPCR.

Luciferase Reporter Assay

To prepare the luciferase-3′ UTR reporter plasmids, 3′UTR fragments fromgenomic DNA extract of hTS cells were amplified. The 3′ UTR PCR fragmentwas cloned into the pGL4.51 vector (Promega, Madison, Wis.) downstreamof the luciferase gene by using PsiI and MfeI (Thermo Scientific,Rockford, Ill.). Primers for 3′ UTR reporter construct were listed asfollowings: For CDX2 3′ UTR region: forward,5′-aaattataagctgtttgggttgttggtct-3′ (SEQ ID NO: 1) and reverse,5′-aaacaattgcccccataatttctgactgc-3 (SEQ ID NO: 2); For SMAD4 3′ UTRregion 1: forward, 5′-aaattataactcccaaagtgctgggatta-3′ (SEQ ID NO: 3)and reverse, 5′-aaacaattgctgcactgttcacaggagga-3 (SEQ ID NO: 4); ForSMAD4 3′ UTR region 2: forward, 5′-aaattataacagttgtcccagtgctgcta-3′ (SEQID NO: 5) and reverse, 5′-aaacaattgatgacttgcccaaaggtcac-3 (SEQ ID NO:6); For GSK3β 3′ UTR region: forward,5′-aaattataacccacaactggggtaaaaga-3′ (SEQ ID NO: 7) and reverse,5′-aaacaattgctgtggaaggggcaaagata-3 (SEQ ID NO: 8).

For dual luciferase assays, firefly luciferase reporter (500 ng) orempty vector without any 3′UTR co-transfected with pGL4.74 and renillaluciferase plasmid (500 ng, Promega), and non-specific control miRNA (30pmol) or miR-124a precursor (30 pmol; System Biosciences, Mountain View,Calif.) were co-transfected to hTS cells (1.5×10⁴ cells in each well)using TransIT®-LT1 transfection reagent (Minis Bio LLC, Madison, Wis.).After transfection (36 hr), the luciferase activity was analyzed by thedual luciferase reporter assay system (Promega) and the Centro LB 960Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).For evaluation, renilla luciferase value was first normalized to thefirefly luciferase activity and the calculated activity of each 3′UTRreporter was further normalized to the control vector. Data representedas mean±SD, n=8, p<0.05 as statistic significance. Whole cell extractsprepared in the cell lysis buffer were subjected to immunoblotting withCDX2, SMAD4, GSK3β, and β-actin antibodies.

Immunofluorescence Staining

Slides with cell culture was fixed for 30 min at room temperature in 95%(v/v) ethanol, washed three times in PBS and incubated with blockingbuffer PBS containing 0.1% (wt/v) Triton X-100 (Sigma) and 5% (v/v)normal donkey serum (Millipore) for 60 min. Primary and secondaryantibodies were diluted in blocking buffer. Primary antibody wasincubated. After incubation with specific primary antibody in PBS at 4°C. (24 hr) or room temperature (2 hr), appropriate fluoresceinisothiocyante (FITC, Invitrogen) or Alexa Fluor 488, 594, and 647(Invitrogen) or Dylight 488 and 594 (BioLegend) conjugated secondaryantibody was added at room temperature (1 hr). After DAPI staining ofnucleus (5 min), incubation with secondary antibody (1 hr) at roomtemperature, and washes, sample was mounted with 50% glycerol. Imageswere captured by confocal laser scanning microscopy (LSM700; Zeiss Z1 orOlympus FluoView 1000 confocal laser scanning microscope) or TissueFAXSsystem (TissueQnostics GmbH, Vienna, Austria). Data were analyzed byTissueQuest software.

Flow Cytometry

After transfection with non-specific shRNA or shRNAs against CDX2, OCT4,SOX2, and NANOG, cells (5×10⁶ cells/ml) were incubated with specificprimary antibodies for 30 min as listed in Table 1. Followed byincubation with the appropriate fluorescent dye-conjugated primaryantibody at adjusted dilution for 1 hr at 4° C., samples were washed andre-suspended in PBS. After passing through polystyrene round-bottom tubewith cell strainer cap (BD Falcon), sample was subjected for flowcytometry (FACScan, BD Biosciences, San Jose, Calif.). Data wereanalyzed with Cell-Quest software (BD Biosciences).

Electron Microscopy

For transmission electron microscopy, the hTS cell-derivedhepatocytes-like cells (at day-4 after induction) were fixed in 0.1 Msodium cacodylate buffer (pH 7.4) containing 3% wt/vol formaldehyde,1.5% (wt/vol) glutaraldehyde and 2.5% (wt/vol) sucrose at roomtemperature (RT) for 1 hr or at 4° C. overnight. The samples were washedwith 0.1 M sodium cacodylate buffer (pH 7.4) before and after osmicationtreatment (2 hr) at 4° C. in Palade's fixative containing 1% (vol/vol)OsO₄. After treated with tannic acid, stained with 1% uranyl acetate,and dehydrated through a graded series of ethanol solutions, sample wasembedded in TABB epoxy resin (Agar Scientific Ltd.). Ultrathin sectionswere stained with uranyl acetate and lead citrate and analyzed by usingJEM-2000 EXII Transmission electron microscope (JEOL, Tokyo).

LDL Uptake Assay

LDL uptake was performed by using LDL Uptake Cell-Based Assay Kit asmanufacturer's instruction (Cayman Chem Co. Ann Arbor, Mich.,). Briefly,5×10⁴ cells were seeded at coverslip in each well of a 24-well plate.hTS cells (as control) and the differentiated hepatocyte-like cells(hHLCs) were fixed after 5 μg/ml LDL-DyLight™ 549 probe treatment (4 hr,37° C.) and then stained for LDL receptor by rabbit anti-LDL andDyLight™ 488-conjugated Goat anti-rabbit antibody. Nuclei werevisualized with DAPI. The final staining was observed by fluorescencemicroscopy.

Oil-O-Red Test

For detection of lipid accumulation, differentiated cells were fixedwith 4% paraformaldehyde (20 min) at room temperature (RT) and washedwith 60% isopropanol for 5 min. After incubation at RT (20 min) with afreshly prepared 60% Oil Red O solution (0.5 g Oil Red O in 100 mlisopropanol passed through a 0.22 μM filter before using, Sigma), cellswere rinsed with 60% isopropanol and counterstained with Hematoxylin I(Thermo Scientific) for microscopy.

Glycogen Storage Test

For glycogen detection, differentiated cells were fixed by 4%paraformaldehyde. Fixed samples were permeabilized with 0.4% TritonX-100. Undifferentiated control cells were incubated with Diastase (1mg/ml in PBS; Sigma) for 1 hr at 37° C. Cells were incubated withperiodic acid (0.5 g dissolved in 100 ml distilled water) for 5 min atRT, washed with distilled water, and incubated with fresh preparedSchiff's reagent (15 min) and subjected for microscopy.

Albumin and Urea Assays

The concentrations of total protein, albumin, and urea in the culturemedium of hTS cells were measured before and after induction by anautomatic analyzer (Hitachi 7080; Tokyo, Japan).

Statistical Analysis

All of the experiments were conducted in triplicate and repeated twotimes as indicated. Data obtained from Western blots, qPCR, luciferasereporter assay, and flow cytometry were calculated by Student's t-test.p-value<0.05 was considered statistically significant.

A Cellular Process from hTS Cells to DE Lineages.

Differentiation of human pluripotent stem cells to hepatocyte-likecells, DE formation is the first step needed to be identified during thecell processes. It was found that in hTS cells, bFGF (10 ng/ml) enabledto efficiently yield DE at 8 hr induction, expressing specificbiomarkers; forkhead box protein A2 (FOXA2) and SRY-box 17 (SOX17),Goosecoid (GSC), and Homeodomain protein MIXL1 by immunofluoresencemicroscopy (FIG. 1A-1C). Subsequently, a timeline expression of theDE-associated markers was established, including transcription factors:GSC, brachyury (T), MIXL1, SOX17, and FOXA2, upon bFGF induction byimmunoblotting assays (FIG. 1D). It was found that levels of MIXL1,Brachyury, and GSC significantly elevated at the initial 15 min,suggesting a transition of primitive streak. These levels, however,declined after 30 min, implicating a migration from primitive streak toa nascent mesendoderm. Specifically, MIXL1 levels decreased from thepeak (15 min) down to a nadir 50% lower than the native one) at 4 hrand, henceforth, the levels returned to the original ones. This fact wassupported by imaging study (FIG. 1C) and TissueFAX analysis (FIG. 7).These results suggested the cellular processes moving from themesendoderm to the DE stage because: i) MIXL1 mRNA expression is absentfrom endoderm but confined to the mesoderm, and ii) loss of MIXL1 has animpact on the endoderm potential of the mesendoderm progenitors as MIXL1is expressed specifically in the primitive streak. SOX17 levelsupregulated to peak at 2 hr (1.5-fold) but downregulated at 8 hr; whilea continually upregulated FOXA2 to the highest levels (3-fold) at 8 hr(FIG. 1D). However, there was a downregulation of GSC from 30 min to 1hr but still sustained at a slightly higher than the original levels(FIG. 1D). To this end, these data suggest that bFGF (10 ng/ml) enablesto induce transdifferentiation of hTS cells to DE lineages in a highlyefficient manner by upregulating SOX17, FOXA2, and GSC, butdownregulating MIXL1 at 8 hr induction. Wherefrom, DE gives rise toepithelial lining of the lungs, esophagus, stomach, and intestines, aswell as endocrine glands such as the liver, pancreas, and thyroid.

Dexamethasone and SOX17 Driving Differentiation to Hepatic Fate by HNF4αExpression

Hepatocyte nuclear factor 4 (HNF4) is an essential transcription factorsfor specification of human hepatic progenitor cells. Since dexamethasone(Dexa) induces HNF4α expression and cytokine oncostatin M (OSM) involvesin the fetal liver development. It was found that by addingdexamethasone (0.1 μM) and oncoststin M (10 ng/ml) to the conditionedculture medium at 8 hr of induction resulted in the differentiation ofhepatocyte-like cells, expressing hepatic markers after 4 days byimmunostainings, for example, albumin, α-fetoprotein (AFP), biliarytransport protein MRP2 (ABCC2), and bile salt export pump (BSEP) (FIG.1E).

Subsequently, immunoblotting assays in time course revealed that bothFOXA2 and SOX17 sustained at a higher level towards 24 hr, following asudden declination at day-2 (FIG. 1F). Interestingly, SOX17 can directlyactivate zinc finger protein 202 (ZFP202) that suppresses HNF4α duringendoderm differentiation. In DE stage, a significant reduction of SOX17levels after peaking (2 hr) (FIG. 1D) that probably indirectly reducedthe suppressive effect on HNF4α; thereby, facilitated thedexamethasone-induced HNF4α activation. As such a combinatory effect ofbFGF, dexamethasone, and SOX17 withdrawal devotes the initiation ofhepatic specified endoderm by HNF4α expression. Moreover, albuminexpressed after 12 hr, supporting a differentiation to hepaticendodermal lineages (FIG. 1F). Consequently, active HNF4α may controlmorphological and functional differentiation of hepatocytes; thereby,the entrance of hepatoblastic stage can take place at day-2 ofinduction. The appearance of AFP at day-3 indicated a cell process offetal immature hepatocytes. Interestingly, a peak level of betatrophinappeared at day-3, suggesting its involvement in the regulation ofnutrition and lipid metabolism. Taken together, hTS cells can beefficiently differentiated to hepatocyte-like cells within week,mimicking cellular processes of primary hepatocytes in embryogenesis.Indeed, these findings suggest that this two-step regimen enables togenerate hepatocyte-like cells from hTS cells distinct from thetime-consuming protocols reported previously in hES cells and iPS cells.

Regulatory Mechanisms of miRNA-124a in DE Specification.

Understanding the molecular mechanisms of how bFGF induces DE formationare essential and required for further hepatic differentiation. bFGFenabled to induce the PI3K/AKT/CREB1 signaling pathway via its receptorFGFR1 (FIG. 8A-8D). Activation of CREB1 therefore allowed us to focus onthe involvement of microRNAs (miRs), the small non-coding RNAs, becauseof two reasons: i) miR-124a enables to regulate FOXA2 in pancreaticβ-cells, the hepatocyte counterpart of DE-derivation and ii) miR-124binding to the site of CREB1 gene is conserved in the mammalian CREB13′UTR. Generally, miRs play as key regulators involved in variousbiological processes including the stem cell differentiation byinhibiting translation and/or to cause RNA degradation. To that, it wasfound that active CREB1 enabled to target at three sites of the promoterof miR-124a mRNA by ChIP-qPCR assay to promote miR-124a expression (FIG.2A). This action was supported by knockdown of CREB1 that reducedmiR-124a expression (FIG. 2B) and, also, a parallel correlation inexpression by qPCR assay (FIG. 2B). Together, these results suggest thatbFGF enables to induce the PI3K/AKT/CREB1 signaling pathway and, inturn, CREB1 promoted miR-124a expression peaking at 4 hr inductionduring DE stage.

Notably, a correlative expression between the highest miR-14a and thelowest MIXL1 at 4 hr induction attracted us to examine whether there wasa relationship between them. By sequence analysis and luciferasereporter assay, miR-124a enabled to target Smad4 messenger RNA (mSmad4)at two sites, resulting in the prevention of signal transduction proteinSmad4 production evidenced by the luciferase reporter assay (FIG. 2D)and immunoblotting assay (FIG. 2E). Consistently, the inhibitory Smad4of miR-124 caused a suppression of MIXL1, supported by knockdown ofSmad4 (FIG. 2F). These data explained the downregulation of MIXL1 in DEstage, which was attributed to the activation of bFGF-induced miR-124a.Furthermore, miR-124a also enabled to play other roles, for example, itsuppressed glycogen synthase kinase 3β (GSK3β) mRNA by luciferasereporter assay (FIG. 2G) that inhibited the production of GSK3β (FIG.2E). This inhibitory GSK3β resulted in the nuclear translocation of itsdownstream substrate β-catenin. In the nucleus, β-catenin enabled totarget the promoter of FOXA2 gene by ChIP-qPCR assay to produce FOXA2(FIG. 2H). To this end, the expression of FOXA2 highlights the celldifferentiation at DE stage.

Maintenance of Self-Renewal Characteristics by OCT4

On the other hand, miR-124a also enabled to target CDX2 mRNA (FIG. 2I)to inhibit its translation that decreased CDX2 production of CDX2, apluripotent transcription factor (FIG. 2E). This was supported by thepresence of co-transfected miR-124a- and CDX2 plasmids (FIG. 2I). Theinhibitory CDX2, however, led to an activation of pluripotenttranscription factor OCT4 (FIG. 2E) via the reciprocal inhibitoryrelationship between OCT4 and CDX2. This function was reflected by theimaging study (FIG. 2J) and immunoblotting assay (FIG. 2K). OCT4activation could be inhibited by anti-miR-124a antibody, linkingmiR-124a and OCT4 by immunoblotting assay (FIG. 2E). The gradualelevation of both pluripotent transcription factors NANOG and SOX2towards 8 hr (FIG. 2K) suggested a supportive role in the maintenance ofpluripotency of DE lineages compatible with that in hES cells.Therefore, these data suggest that OCT4 mainly maintains theself-renewal characteristics of DE, supported by other core pluripotencytranscription factors NANOG and SOX2. Furthermore, the active OCT4 wascapable of targeting at two sites of the promoter of SOX17 mRNA byChIP-qPCR assay (FIG. 2L) that induced SOX17 expression peaking at 2 hr(FIG. 1C). SOX17 expression represents another milestone in DEidentification. Taken together, these results demonstrate in hTS cellsthat bFGF-dependent miR-124a induces DE formation in a highly efficientmanner (8 hr) distinct from the 3-day course in hES cells. A schematicregulatory molecular mechanism illustrates the DE specification throughthe miR-124a signaling to upregulate FOXA2, SOX17, and OCT4 butdownregulate MIXL1; wherein OCT4 plays a main role in the maintenance ofself-renewal of DE lineages (FIG. 2L).

Genetic Profiles Correspond with the Stage-Specific Phenotypes inHepatic Development

To subsequently direct DE differentiation into hepatic lineages,dexamethasone and oncostatin M were added after completion of DE stageat 8 hr. Given the advantages of qRT-PCR assay, the dynamic expressionof hepatic development-associated 31 genes in a time course profile wasexplore to characterize mRNA fingerprint that could be used to followthe differentiation process. In all, the genetic profiles of each generevealed a similar pattern with 4-5 peaks during 6-day induction. Basedon the cellular processes of hepatic development in mouse model, the hTScells treated with bFGF and the cocktail of dexamethasone and oncostatinM were classified based on their genetic expression profiles into fourstages of cellular processes: primitive streak to DE (<8 hr), hepaticspecified endoderm lineages (8 hr to day-1), hepatoblasts (day 2-4), andfetal and adult hepatocyte-like cells (day≧day 4) (FIG. 9A-9F).

Therefore, these results revealed that i) at the stage of primitivestreak to DE, four genes expressed predominantly (>10- to 1,000-fold),include CXCR4, FOXA2, SOX17, and HHEX; ii) at the hepatic specifiedendoderm, six genes expressed predominantly (between 10- and 100-fold),including SOX17 for liver bud formation and lipid metabolism, thyroxin-and retinol-binding protein TTR, proteins carrier albumin (ALB),tyrosine catabolism enzyme TAT, SERPINA1, and bile acid biosynthesisenzyme CYP7A1; iii) at the hepatoblastic stage, there were 6 genesexpressed predominantly (between 10- and 100-fold), including TTR, ALB,TAT, CYP7A1, SERPINA1, and bile salts excretion pump BSEP; and iv) atthe fetal and adult hepatocyte-like cell stage, there were 11 genesexpressed prominently (>100-fold), including HHEX, BSEP, TTR, ALB, TAT,SERPINA1, glucose homeostasis enzyme G6PC, hepatobiliary excretiontransporter MRP2 (ABCC2), immune and normal macrophage regulator C/EBPβ,and several hepatic gene regulators such as HNF4α and HNF1α.Furthermore, expression of α-fetoprotein (AFP) appeared at fetal stage(day-4 and day-5), but decreased after day-5 of adult hepatocyte-likecell stage (FIG. 6).

Hepatic Plate-Like Architecture Exhibits Signatures of Hepatocytes

In liver, hepatic plate system constitutes a unique tissue structure,composed of bile ducts and blood vessels surrounded by a sheath that iscontinuous with Glisson's capsule. To characterize the morphologicchanges of hTS cell-derived hepatocyte-like cells duringdifferentiation, it was found that cellular morphology changed from theinitial fibroblast-like to a longer spindle feature in accompany with atrend to form lobular shape, showing numerous polygonal cells localizedat the central areas at day-2 and day-3 (FIG. 3A). After day-4, however,cells might aggregate to form a plate-like mass and this phenomenondepended on the initial cell number cultured. The cell mass was thensubjected to further examinations.

Histologically, most of the cellular arrangement appeared to be one cellor two cells thick eosinophilic cytoplasm to form a plate-like tissue;however, cells might aggregate together to form a cell mass (FIG. 3A).Immunohistochemically, these cells exhibited immunofluorescencestainings for Albumin, AFP, Betatrophin, HNF4α, APOF, CPS1, ADH1, andCYP2B6 in the cytoplasmic compartment; while a subset of cell membranemarkers, including CXCR4, CX32, MRP2, and BSEP, making the cell inpolygonal shape similar to the primary hepatocytes (FIG. 3B). Electronmicroscopy displayed ultrastructures similar to the primary hepatocyte,for example, large cytoplasm to nucleus ratio, plenty of mitochondoria,well-organized endoplasmic reticulum, desmosome junction, intact golgiapparatus, and specifically, the enlarged lumen of the canaliculus andthe junctional complexes (FIG. 3C). Moreover, immunocytofluoresenceimaging revealed a colocalization of AFP and Albumin as well as ABCC2(MRP2) and BSEP (FIG. 1E). To this end, it was demonstrated that thesehepatocyte-like cells possess hepatic characteristics of either cellularcomponents or infrastructures that resemble to the primary hepatocytes.

Hepatocyte-Like Cells Function as Primary Hepatocytes In Vitro and InVivo

The liver is the most important organ responsible for many specificfunctions in metabolisms such as albumin synthesis, ureagenesis,glycogenesis, and detoxification. To examine whether thesehepatocyte-like cells are able to function similar to the primaryhepatocytes, the secreting capability of albumin and urea in the cellswere investigated. Culture medium at day-4 induction was harvested andsubjected to the ELISA analysis. The results revealed that both albuminand urea levels were significantly increased, suggesting theircapabilities to produce secreting albumin and urea in the culture medium(FIG. 4A). Next, glycogen storage capacity test revealed a positiveperiodic acid-Schiff (PAS) staining after diastase digestion treatmentin either cellular phase or hepatic plate-like tissue (FIG. 4B). Thisaction was further confirmed by the PAS fluorescence emission for bothimmunocytochemistry and immunohistochemistry (FIG. 4B). In addition, LDLuptake assay revealed that the ability of LDL uptake of cells initiatedat the immature hepatocytes of day-4 induction (FIG. 4C). Oil-O-Redstaining revealed the presence of lipid droplets, suggesting somehow thedegree of adipogenesis (FIG. 4D). Furthermore, it was examined whetherthe hepatocyte-like cells possess the capacity of drug metabolism anddetoxification in vitro by using cytochromes P450 (CYPs) enzymes astarget by qPCR analysis. The results revealed that the hepatocyte-likecells significantly expressed activities of CYP1A2, CYP2B6, CYP2C8,CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP7A1 in responsive to metabolizespecific drugs (FIG. 5A-5I), suggesting that these hepatocyte-like cellscan be used for drug screening and discovery similar to primaryhepatocytes. For example, a liver enzyme-inducer rifampin can promoteCYP3A4 activity to increase the metabolic rate of drug and inhibition ofCYP3A4 is a major cause of drug-drug interactions (DDI), which have beenwidely used in the clinical settings. Furthermore, it has been shownthat phloracetophenone (2,4,6-trihydroxyacetophenone, THA) promotes bothCYP7A1 activity and mRNA expression to reduce both plasma cholesteroland triglyceride in hypercholesterolemic hamsters and THA antagonizedthe inhibitory regulation of chenodeoxycholic acid (CDCA) on CYP7A1 mRNAexpression. To that, it was shown that in the hepatocyte-like cells,refampin-induced CYP3A4 was able to be reduced by its inhibitoritraconazole (FIG. 5H), while THA induced elevation of CYP7A1 mRNA wasreduced by CDCA (FIG. 5I). From the pharmacological point of view, forexample, bile acid binding resins are indicated for the treatment ofelevated plasma low-density lipoprotein cholesterol concentrations,however, resin therapy is hazardous. Therefore, new drugs have beendesigned by approaching the regulation of CYP7A1 to reduce plasmacholesterol and be based on the confirmation of CYP7A1 position as afocus for innovative pharmacological intervention.

Basic FGF Induced Activation of PI3K/AKT/CREB1 Signaling Pathway in hTSCells

The hTS cells were treated with bFGF (10 ng/ml) in the conditionedmedium, and it was shown that FGF receptor (FGFR) inhibitor PD166866could block the bFGF-induced activation of phosphatidylinositol 3-kinase(PI3K) by immunoblotting assay (FIG. 8A), suggesting that the inhibitoryeffect was through the FGFR at the cell membrane. As a result, thedownstream effector AKT was phosphorylated evidenced by using PI3K siRNA(FIG. 8B). To clarify which protein kinase B (AKT) subunit wasactivated, specific siRNA against three AKT subunits: AKT1, AKT2, andAKT3 were examined by immunoblotting assay. The result showed that onlyAKT1 phosphorylated and activated its downstream effector cAMP responseelement-binding protein 1 (CREB1) (FIG. 8C). This function was furtherconfirmed by immunoprecipitation (IP) assay (FIG. 8D). Taken together,this indicates that bFGF induces activation of the PI3K/AKT1/CREB1signaling pathway at 4 hr induction.

Example 2 Experimental Procedures Cell Culture and Differentiation

This study was approved by the Institutional Review Board on HumanSubjects Research and Ethics Committees (KMUHIRB-20140071). The hTScells were obtained with informed consent as described previously (Leeet al., 2012, PLoS ONE 7, e52491) and maintained in α-MEM (Gibco) mediumsupplemented with 10% (v/v) fetal bovine serum (FBS; SAFC Biosciences)at 37° C. in humidified air containing 5% CO₂. For DE differentiation,cells were carried out by a conditioned α-MEM media containing 10% FBS,2-mercaptoethanol (1 mM), nicotinamide (10 mM), and bFGF (10 ng/ml) for8 hr, according to the empirical studies (data not shown). Forhepatocyte differentiation after DE formation at 8 hr, cell culture waschanged to medium containing bFGF (10 ng/ml), dexamethasone (0.1 μM,Sigma), recombinant human oncostatin M (10 ng/ml, Excel-BiomedicalInc.), BMP4 (20 ng/ml), and HGF (5 ng/ml). Cells were harvested at 4-7days for assay as indicated. Determination of stage-specificdifferentiation of lineages depends on the hepatic cell-associatedmarkers in liver development.

Animal Study

Adult male Sprague-Dawley rats (300-350 g) were housed in a 12 hrlight/12 hr dark cycle with ad libtum access to food and water.Experimental studies were approved by Institutional Animal EthicalCommittee (IAEC) of Kaohsiung Medical University (IACUC-96009). Forexperiments, rats were anesthetized with chloral hydrate 25% (500 mg/kg)via intraperitoneal injection. Rats were divided into two groups: thesham operation as control (n=8) and the other as study group (n=8).Intravenous blood (0.5 ml) was obtained as baseline before experimentand both groups were injected intraperitoneally with CCl4 (1 ml/kg: 1:1v/v in corn oil) after 12 hr. Immediately, study group was injected byhTS cell-derived hepatocytes (1×10⁶ cells/200 μl culture medium) fromthe tail vein and sham group was given PBS solution only. Serum samplewas taken at baseline, cell injection point, 24 hr, 48 hr, and 72 hr andsubjected for liver function tests, namely, aspartate aminotransferase(AST or formerly called SGOT), alanine aminotransferase (ALT or formerlycalled SGPT), and alkaline phosphatase (ALP), serum bilirubin. All ratswere sacrificed at day 4 to obtained liver and lung organs forhistopathological studies.

mRNA, miRNA, Chromatin Immunoprecipitation (ChIP)-qPCR, and mRNAMicroarray

Methods were performed as described previously (Lee et al., 2012, PLoSONE 7, e52491). For mRNA expression, RNA was isolated from hTS cells intriplicate or quintuple samples using TRIZOL reagent (Invitrogen) withDNAase I on-column digestion (Qiagen, Valencia, Calif.) according tomanufacturer's instruction. Total RNA (500 ng) was used for reversetranscription with iScript cDNA synthesis kit (Bio-Rad). Real-timepolymerase chain reaction (qPCR) carried out in duplicate using1/40^(th) of the cDNA per reaction and 400 nM forward and reverseprimers. Comparative real-time PCR was carried out at least triplicateusing the Power SYBR® Master Mix (Applied BioSystems) with the 7500Real-Time PCR System (Applied Biosystems). All genes were normalized tothe GAPDH expression and were normalized to the expression ofundifferentiated hTS cells using the ΔΔCt method unless statedotherwise. Primer sequences used in this study can be found in Table 5.

For miRNA analysis, 25 ng of total RNA was reverse-transcribed using theTaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qPCR wascarried out at least triplicate using the TaqMan Universal PCR MasterMix (Applied Biosystems) with the 7500 Real-Time PCR System (AppliedBiosystems) including no-template controls, using specific primers formiR-124a or RNU6B (Applied Biosystems). U6 snRNA (RNU6B; AppliedBiosystems) served as an endogenous control.

For ChIP assay, hTS cell samples of indicated time in induction werefixed with a final concentration of 1% formaldehyde. After incubation atroom temperature (10 min), the reaction was stopped by adding glycine(125 mM). ChIP assay was performed using a protocol associated with theChIP assay kit (Upstate Biotechnology). After extensive washing, ChIPedDNA was eluted from the beads and analyzed by qPCR.

Luciferase Reporter Assay

To prepare the luciferase-3′ UTR reporter plasmids, amplified were 3′UTRfragments from genomic DNA extract of hTS cells. The 3′ UTR PCR fragmentwas cloned into the pGL4.51 vector (Promega, Madison, Wis.) downstreamof the luciferase gene by using PsiI and MfeI (Thermo Scientific,Rockford, Ill.). Primers for 3′ UTR reporter construct were listed asfollowings:

For Cdx2 3′ UTR region: forward, (SEQ ID NO: 1)5′-aaattataagctgtttgggttgttggtct-3′ and reverse, (SEQ ID NO: 2)5′-aaacaattgcccccataatttctgactgc-3; For Smad4 3′ UTR region 1: forward,(SEQ ID NO: 3) 5′-aaattataactcccaaagtgctgggatta-3′ and reverse,(SEQ ID NO: 4) 5′-aaacaattgctgcactgttcacaggagga-3; For Smad4 3′UTR region 2: forward, (SEQ ID NO: 5)5′-aaattataacagttgtcccagtgctgcta-3′ and reverse, (SEQ ID NO: 6)5′-aaacaattgatgacttgcccaaaggtcac-3; For GSK3β 3′ UTR region: forward,(SEQ ID NO: 7) 5′-aaattataacccacaactggggtaaaaga-3′ and reverse,(SEQ ID NO: 8) 5′-aaacaattgctgtggaaggggcaaagata-3.

For dual luciferase assays, firefly luciferase reporter (500 ng) orempty vector without any 3′UTR co-transfected with pGL4.74 and renillaluciferase plasmid (500 ng, Promega), and non-specific control miRNA (30pmol) or miR-124a precursor (30 pmol; System Biosciences, Mountain View,Calif.) were co-transfected to hTS cells (1.5×10⁴ cells in each well)using TransIT®-LT1 transfection reagent (Minis Bio LLC, Madison, Wis.).After transfection (36 hr), the luciferase activity was analyzed by thedual luciferase reporter assay system (Promega) and the Centro LB 960Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).For evaluation, renilla luciferase value was first normalized to thefirefly luciferase activity and the calculated activity of each 3′UTRreporter was further normalized to the control vector. Data representedas mean±SD, n=8, p<0.05 as statistic significance. Whole cell extractsprepared in the cell lysis buffer were subjected to immunoblotting withCdx2, Smad4, GSK3β, and β-actin antibodies.

Plasmids

MiR-124a precursor and anti-miR-124a were purchased from SystemBiosciences. Briefly, miR-124a precursor (60 pmol) or anti-miR-124a (60pmol) was transfected to hTS cells in 12-well culture dishes usingTransIT-LT1 transfection reagent (Minis, Madison, Wis.). Total RNAs wereused for quantifying miR-124a at 36 hr after transfection. Smallinterfering RNA (siRNA) targeting PI3K (SASI_Hs01_00233971 andSASI_Hs01_00127787), Akt1 (SASI_Hs01_00205545), and Akt2(SASI_Hs01_00035055) were purchased from Sigma. Short hairpin RNA(shRNA) targeting CREB1 (TRCN0000007310, TRCN0000226467 andTRCN0000226468), Smad4 (TRCN0000010321, TRCN0000010323 andTRCN0000040032), Akt3 (TRCN0000001615 and TRCN0000001616), Oct4(TRCN0000004879 and TRCN0000004882), Cdx2 (TRCN0000013683 andTRCN0000013686), and control shRNA (shGFP; TRCN0000072178,TRCN0000072179 and TRCN0000072183) were purchased from National RNAiCore platform, Academia Sinica, Taiwan. Transfection was performed withsiRNA or shRNA at 2 μg plus 4 μl transfection reagent.

LDL Uptake Assay

LDL uptake was performed by using LDL Uptake Cell-Based Assay Kit asmanufacturer's instruction (Cayman Chem Co. Ann Arbor, Mich.,). Briefly,5×10⁴ cells were seeded at coverslip in each well of a 24-well plate.hTS cells (as control) and the differentiated hepatocyte-like cells(hHLCs) were fixed after 5 μg/ml LDL-DyLight™ 549 probe treatment (4 hr,37° C.) and then stained for LDL receptor by rabbit anti-LDL andDyLight™ 488-conjugated Goat anti-rabbit antibody. Nuclei werevisualized with DAPI. The final staining was observed by fluorescencemicroscopy.

Oil-O-Red Test

For detection of lipid accumulation, differentiated cells were fixedwith 4% paraformaldehyde (20 min) at RT and washed with 60% isopropanolfor 5 min. After incubation at RT (20 min) with a freshly prepared 60%Oil Red O solution (0.5 g Oil Red O in 100 ml isopropanol passed througha 0.22 μM filter before using, Sigma), cells were rinsed with 60%isopropanol and counterstained with Hematoxylin I (Thermo Scientific)for microscopy.

Glycogen Storage Test

For glycogen detection, differentiated cells were fixed by 4%paraformaldehyde. Fixed samples were permeabilized with 0.4% TritonX-100. Undifferentiated control cells were incubated with Diastase (1mg/ml in PBS; Sigma) for 1 hr at 37° C. Cells were incubated withperiodic acid (0.5 g dissolved in 100 ml distilled water) for 5 min atRT, washed with distilled water, and incubated with fresh preparedSchiff's reagent (15 min) and subjected for microscopy.

Biochemical Parameter Tests

All biochemical parameters of liver function including albumin, urea,aspartate aminotransferase (AST), and alanine aminotransferase (ALT)were measured by using auto-analyzer (Hitachi 7080, Japan).

Functional Cytochrome p450 Assay

To test the activity of CYP enzyme induction in hTS cell-derivedhepatocyte-like cells, cells were treated with reagents for 24 hr, forexample, for CYP1A2 test, used were rifampicin (25μM)/rifampicin+ciprofloxacin (1 μM); for CYP2B6 test by phenobarbital(100 μM)/phenobarbital+clopidogrel (25 μM), for CYP3A4 by rifampicin (25μM)/rifampicin+itraconazole (25 μM), for CYP7A1 by2,4,6-trihydroxyacetophenone (THA, 1 μM)/THA+CDCA (25 μM), for CYP2C8and CYP2C9 by rifampicin (25 μM)/rifampicin+gemfibrozil (25 μM) and forCYP2C19 by rifampicin (25 μM)/rifampicin+ticlopidine (25 μM). Huh-7cells were used as positive control.

Immunocytochemistry and Immunohistochemistry

Methods were performed as described previously (Lee et al., 2012, PLoSONE 7, e52491). Briefly, slides with cell culture was fixed for 30 minat room temperature in 95% (v/v) ethanol, washed three times in PBS andincubated with blocking buffer PBS containing 0.1% (wt/v) Triton X-100(Sigma) and 5% (v/v) normal donkey serum (Millipore) for 60 min. Primaryand secondary antibodies were diluted in blocking buffer. Primaryantibody was incubated. After incubation with specific primary antibodyin PBS at 4° C. (24 hr) or room temperature (2 hr), appropriatefluorescein isothiocyante (FITC, Invitrogen) or Alexa Fluor 488, 594,and 647 (Invitrogen) or Dylight 488 and 594 (BioLegend) conjugatedsecondary antibody was added at room temperature (1 hr). After DAPIstaining of nucleus (5 min), incubation with secondary antibody (1 hr)at room temperature, and washes, sample was mounted with 50% glycerol.Images were captured by confocal laser scanning microscopy (LSM700;Zeiss Z1 or Olympus FluoView 1000 confocal laser scanning microscope) orTissueFAXS system (TissueQnostics GmbH, Vienna, Austria). Data wereanalyzed by TissueQuest software.

Electron Microscopy

For transmission electron microscopy, methods were performed asdescribed previously (Lee et al., 2012, PLoS ONE 7, e52491). Briefly,the hTS cell-derived hepatocytes-like cells (at day-4 induction) werefixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 3% wt/volformaldehyde, 1.5% (wt/vol) glutaraldehyde and 2.5% (wt/vol) sucrose atRT for 1 hr or at 4° C. overnight. The samples were washed with 0.1 Msodium cacodylate buffer (pH 7.4) before and after osmication treatment(2 hr) at 4° C. in Palade's fixative containing 1% (vol/vol) OsO₄. Aftertreated with tannic acid, stained with 1% uranyl acetate, and dehydratedthrough a graded series of ethanol solutions, sample was embedded inTABB epoxy resin (Agar Scientific Ltd.). Ultrathin sections were stainedwith uranyl acetate and lead citrate and analyzed by using JEM-2000 EXITTransmission electron microscope (JEOL, Tokyo).

Immunoblotting and Immunoprecipitation (IP)

Methods were performed as described previously (Lee et al., 2012, PLoSONE 7, e52491). For immunoblotting assay, cells were harvested into RIPAlysis solution (Millipore, Billerica, Mass.) supplemented with proteaseand phosphatase inhibitors (Roche). After electrophoresis of 30 μglysates on polyacrylamide gels, electroblotting onto PDVF membranes(Millipore) was performed. After blocked by 5% non-fat milk in PBS atroom temperature (1 hr), target protein was detected by using primaryantibody. All membranes were incubated with chemiluminescent (Millipore)and imaging was captured by the ChemiDoc XRS system (Bio-RAD).Antibodies used were listed in Table 4. Data were analyzed byAlphaEaseFC (version 4.0.0) system. For IP assay, Cell lysates ofbFGF-treated hTS cells were collected. By incubation with proteinG-agarose (Millipore) for 30 min, total protein (100 μg) was treatedwith specific primary antibody overnight listed in Table 4. Aftertreating with protein G-agarose beads (2 hr), sample was washed threetimes with RIPA lysis buffer (Millipore), following by adding withprotein loading dye and boiled for 5 min. The sample was resolved by 8%SDS-PAGE and subjected to immunoblotting analysis.

Sample Preparation for 2-Dimensional Gel Electrophoresis (2-DE)

Two samples were obtained: cell culture medium (5-days, as study group)and pure culture medium (as control group). Samples were prepared asdescribed previously (Chou et al., 2015). Briefly, samples (0.1 ml) wereincubated with 1 ml ice-cold acetone containing 11% trichloroacetic acid(TCA, w/v) and 20 mM DTT for 30 min at −20° C. After centrifugation(12,000 rpm, 10 min, 4° C.), the protein pellet was washed twice with 1ml cold acetone containing 20 mM DTT, followed by air-dry to remove theacetone. Then, appropriate rehydration buffer (7M urea, 2M thiourea, 2%CHAPS, 0.5% IPG buffer, 20 mM DTT) was added and the concentrated samplewas measured by the Bradford method. For 2-DE analysis, a total of 150μg protein was incubated with buffer containing 5M urea, 2M thiourea, 3%w/v CHAPS, 1% immobilized pH gradient (IPG) with a nonlinear pH of 3-10,100 mM DeStreak reagent, and a trace of bromophenol blue. After a seriesof treating processes, the sample was cup-loaded near the anode of theIPG strips using the Ettan IPGphor cup-loading (Amersham Biosciences)and protein focusing was achieved using the IEF parameters according tothe manufacturer's protocol. After first dimensional electrophoresis,isoelectric focusing IPG strips was shaked in a conditionedequilibration buffer containing 1% w/v DTT (15 min), and followed by thesame solution containing 2.5% w/v iodoacetamide (15 min). The strip wastransferred on top of the 12% SDS-polyacrylamide gel (PAGE). The seconddimension separation was performed by a constant 75 V (30 min) and 100 V(16 hr). The 2-DE gel was silver-stained and detected with the Typhoon9410 scanner (Amersham Biosciences) The spots were compared andquantified by using the Image Master 2D Platinum system. (AmershamBiosciences).

Electrospray Ionization-Quadrupole-Time of Flight Tandem MassSpectrometry (ESI-Q-TOF-MS/MS) for Protein Identification andQuantitation

To identify the 2D gel protein observed, the samples were digested bytrypsin, followed by subjecting to the nanoflow liquid chromatographyand Waters-Micromass ESI-Q-TOF for protein identification (Waters,Manchester, UK) as described previously (Chou et al., 2015). To obtainthe corresponding peak lists, the MS/MS spectra of individual fragmentsof each of the precursors were processed by MassLynx 4.0 software(Manchester, UK) and the peak list files were uploaded to an in-houseMascot server for protein identification.

Statistical Analysis

All of the experiments were conducted in triplicate and repeated twotimes as indicated. Data obtained from Western blots, qPCR, luciferasereporter assay, and flow cytometry were calculated by Student's t-test.In animal study, paired ANOVA test was use statistically. p-value<0.05was considered statistically significant.

Results

1) bFGF Alone Induces DE Differentiation in hTS Cells

The path from stem cells to hepatic lineages composes of a progressiveseries of cellular processes, particularly including the required andessential step in DE formation. hTS cells were treated with bFGF (10ng/ml) initially and measured the levels of DE-associated markers overtime by immunoblotting assay. The results showed that at the initial 15min of induction, transcription factors such as goosecoid (Gsc),Brachyury (T), homeodomain protein Mixl1 (Mixl1), SRY-box 17 (Sox17),forkhead box protein A2 (Foxa2, also known as Hnf3β), and the primitiveendoderm marker Sox7 were significantly upregulated, peaking in between30 min and 1 hr (FIG. 10A). These data implicated a fast transition fromhTS cells to the nascent mesendoderm mediating primitive streak stagecompatible with the liver development in early embryogenesis.Henceforth, Sox17 levels continually elevated to 4 hr and declined;whilst Foxa2 and Brachyury elevated to 8 hr but Sox7 expression becamenascent after 15 min of induction. Notably, intensity of Mixl1downregulated from the peak (15 min) to a nadir at 4 hr (˜50% lower thanthe native one) and returned to the original levels at 8 hr measured byTissueFAX analysis (FIG. 10E). Their changes in expression were alsodemonstrated by immunofluoresence imagings (FIG. 10B). These resultsindicate that bFGF alone enables to rapidly differentiate hTS cells toDE stage through primitive streak and mesendoderm mimicking theembryonic liver development.

2) PI3K/Akt/CREB1 Signaling Pathway Promotes MiR-124a Expression

bFGF enabled to induce the PI3K/Akt/CREB1 signaling pathway via itsreceptor FGFR1 in hTS cells. hTS cells were treated with bFGF (10 ng/ml)in the conditioned medium. FGF receptor (FGFR) inhibitor PD166866 couldblock the bFGF-induced activation of phosphatidylinositol 3-kinase(PI3K) by immunoblotting assay (FIG. 10F), suggesting that theinhibitory effect was through the FGFR at the cell membrane. As aresult, the downstream effector AKT was phosphorylated evidenced byusing PI3K siRNA (FIG. 10G). To clarify which protein kinase B (AKT)subunit was activated, specific siRNA against three AKT subunits: AKT1,AKT2, and AKT3 were examined by immunoblotting assay. The result showedthat only AKT1 phosphorylated and activated its downstream effector cAMPresponse element-binding protein 1 (CREB1) (FIG. 10H). This function wasfurther confirmed by immunoprecipitation (IP) assay (FIG. 10I). Takentogether, this indicates that bFGF induces activation of thePI3K/AKT1/CREB1 signaling pathway at 4 hr induction.

microRNA (miR)-124, a small non-coding RNA, is involved in the Foxa2expression in pancreatic β-cells, a derivative of ventral foregutendoderm. Subsequently, in the nucleus, the activated CREB1 directlytargeted at three sites of the promoter of miR-124a to induce miR-124aexpression at 4 hr induction by ChIP-qPCR assay (FIG. 10C) and knockdownof CREB1 reduced its expression (FIG. 10J). Expression of CREB1 andmiR-124 over time appeared in a parallel correlation by qPCR analysis(FIG. 10D). These results indicate that bFGF-induced PI3K/Akt/CREB1signaling pathway enables to spatiotemporally upregulate miR-124a at theearly differentiation of hTS cells.

3) MiR-124a Directs DE Specification

Several genes relevant to hepatogenesis were screened and constructedthe luciferase reporter assays, by which several signal transductionproteins were measured, including mothers against decapentaplegichomolog 4 (Smad4) (FIG. 11A), glycogen synthase kinase 3β (GSK3β) (FIG.11B), and homeobox transcription factor Cdx2 (FIG. 11C). Inhibitoryfunctions of miR-124a include: i) to target at the promoter of Smad4messenger RNA (Smad4 mRNA) to prevent Smad4 production (FIG. 11A,lower). Consequently, the inhibitory Smad4 caused suppression of Mixl1,which was verified by knockdown of Smad4 (FIG. 11J). This mechanismexplained the downregulation of Mixl1 in the DE stage (FIG. 10A) and themigratory cell fate transition during gastrulation; ii) to target at thepromoter of GSK3β mRNA to inhibit its translation (FIG. 11B, lower),thereby, resulting in the nuclear translocation of downstream substratecadherin-associated protein β-1 (β-catenin). In the nucleus, β-catenintargeted the promoter of Foxa2 gene to produce Foxa2 (FIG. 11D),highlighting the differentiation at the stage of DE. Interestingly, thisincreased Foxa2 in turn induced c19orf80 gene transcription, encodingbetatrophin protein expression (FIG. 11E). Betatrophin is a hormoneproduced in liver, controls pancreatic β cell proliferation; iii) totarget at the caudal-related homeobox transcripts Cdx2 mRNA to inhibitits translation to the pluripotent transcription factor Cdx2 (FIG. 11C,lower). All these molecular events occurred at 4 hr induction, which wasconfirmed by using miR-124a and anti-miR-124a antibody by immunoblottingassay (FIG. 11F).

Subsequently, the decreased Cdx2 promoted upregulation of pluripotenttranscription factor Oct4 by immunofluoresence imaging study (FIG. 11G).Overexpression of Oct4 was further verified by knockdown of miR-124ausing anti-miR-124a antibody by immunoblotting assay (FIG. 11F). Thisreciprocal inhibitory relationship between Cdx2 and Oct4 is evidenced byimmunoblotting assay (FIG. 11K, upper). Furthermore, observed was thesignificantly gradual elevation of pluripotency transcription factorNanog at 8 hr induction (FIG. 11K, lower). These results suggested thatOct4 played the main role in maintaining the pluripotent characteristicsof DE lineages; while Nanog might play as a supportive role consistentwith that in hES cells. Importantly, the activated Oct4 in turn targetedat the promoter of Sox17 gene by ChIP-qPCR assay (FIG. 11H) thatpromoted Sox17 expression (FIG. 10A). Expression of Sox17 representedanother milestone in the DE differentiation. Together, FIG. 11I is aschematic illustration to describe the regulatory molecular mechanisms,by which bFGF induction initiated DE formation mediating miR-124a in hTScells.

4) Generation of Hepatocyte-Like Cells in 3-D Tissue Structure

Subsequently, cells were cultured with a combination of bFGF (10 ng/ml),dexamethasone (Dexa; 0.1 μM), oncoststin M (OSM; 10 ng/ml), bonemorphogenetic protein 4 (BMP4; 20 ng/ml), and hepatic growth factor(HGF; 5 ng/ml) after DE formation (8 hr). Unexpectedly, cellularmorphology might exhibit as dispersed fibroblast-like cells or graduallyaggregate to form a crescent cell mass, depending on seeding density inculture (FIG. 12A. insert and FIG. 12F). Histologic examination of thecell mass revealed two distinct peripheral and central compartments,constructing a 3-dimensional (3D) tissue structure. In the peripheralpart, numerous clustered small cells distributed irregularly among theextracellular matrix (ECM) beyond the basement membrane. Cells hadcondensed nuclei, frequently eccentric located, and abundant granularand vacuoles in the eosinophilic cytoplasm similar to the embryonicstem/progenitor cells (FIG. 12F). In the central part, many independentcolumnar ECMs, by cell linings at both sides, distributed from the basaltowards the central areas (FIG. 12A). These cells contained abundanteosinophilic cytoplasm and dispersed chromatin in the single roundnucleus with one or two prominent nucleoli mimicking the phenotypichepatocytes. Several binucleate cells could be seen. This feature issimilar to that known as hepatic plates in human liver.

Immunocytochemically, these hepatocyte-like cells exhibited specificmarker(s) of: i) human cytoplasmic marker stem 121™ for human cells,mast/stem cell growth factor receptor C-kit for liver intrinsic stemcells, CK19 for cholangiocytes, and CK18 for hepatocytes (FIG. 12B); andii) albumin (ALB), α-fetoprotein (AFP), Betatrophin, ADH1, APOF, CPS1,GATA4, CYP1A1, and CYP2B6 in the cytoplasm for hepatocytesimmunohistochemically (FIG. 12C, and FIG. 12E). Whilst a subset ofsurface markers including ASGR1, CXCR4, BSEP, MRP2, and Cx32 constructeda polygonal cell shape similar to the primary human hepatocyte (FIG.12C). Furthermore, electron microscopy revealed a similar ultrastructureto primary hepatocyte, including a large cytoplasm to nucleus ratio,plenty of mitochondoria, well-organized endoplasmic reticulum, tightjunction, numerous lipid vacuoles, glycogen storage, enlarged lumen ofthe bile canaliculus with junctional complexes, and multiplex ECMs (FIG.12D).

5) TGFβ1 Contributes to the Formation of Fibronectin and Collagen IVScaffold in Hepatic ECMs

Among 9 newly upregulated, secreted proteins in the cell-culturedmedium, protein (no. 413) became an attractive target because itsignificantly predicted, by 46% of peptide sequences matched, to be thetransforming growth factor-β (TGF-β)-induced protein ig-h3 precursor(TGFβ1) by Mascot MS/MS ions search system (ESI-QUAD-TOF, Bruker ImpactHD, Matrix Science, USA) (FIG. 13A, red arrow; FIG. 13C). TGFβ1 is amajor fibrogenic, multifunctional cytokine, acting as both autocrine andparacrine manner to enhance fibronectin and collagen formation inhepatic stellate cells (HSCs). Accordingly, to identify the presence ofTGFβ1, immunohistochemistry was used to demonstrate the coexpression ofimmunoreactive TGFβ1, fibronectin, and collagen IV in the ECMs byimmunohistochemistry (FIG. 13B). These results suggest that TGFβ1,fibronectin, and collagen IV constitute, at least partly, the scaffoldof ECMs in the 3-D tissue structure of hepatocyte-like cells that maysupport proliferation and differentiation of hepatocytes in the hepaticplates.

6) Transcriptional Expression Characterizes the Stage-Specific HepaticDifferentiation

Forty two hepatic development-associated genes were analyzed, suggestingthat differentiating cells might share an overlapping pattern in geneexpressions during cellular processes. As mentioned, the transition frompluripotent hTS cells to primitive streak (15 to 30 min) and mesendoderm(<1 hr), expressing an elevation of GSC, Brachyury (T), and Sox7 alongwith a gradual increase of Mixl1, Foxa2 and Sox17 was observed (FIG.10A). Sox7 is primarily expressed in the primitive streak, visceralendoderm, and parietal endoderm but not DE (Kanai-Azuma et al., 2002).At the DE stage (1 to 8 hr), persistence of high transcriptionalexpressions sustained, including CXCR4, Foxa2, Sox17, HHEX, and Sox7,and declined thereafter (Table 3). As cell process entered the hepaticendoderm stage (8 hr to 1 day), a core group of endoderm transcriptionfactors including Sox/7 and Foxa2, in turn, regulated a cascade of genescommitting cells to the endoderm lineage. Shortly after hepaticspecification, the epithelium begins to express genes associated withliver bud genes including Albumin, AFP, and Hnf4α. A large number ofhepatoblast-associated gene expressions emerged to commit celldifferentiation to bipotential hepatoblasts (day-2 to day-4). Wherefromhepatoblasts expressed specific genes in association with fetalhepatocytes (like AFP), adult hepatocytes (like ALB and Hnf4α), andbiliary epithelial cells (like cytokeratin-19), allowing thedifferentiation to reach the fetal/adult hepatocytic stage (>day-4).Together, these progressive transcriptional expressions are consistentwith the cellular processes in liver development as listed in Table 3.

7) Hepatocyte-Like Cells Exhibit Liver Functions

Preservation of liver functions is essential requirement in thepluripotent stem cell-derived hepatocytes. To that, cell culture mediumwas collected and subjected to the enzyme-linked immunosorbant assay(ELISA). The results demonstrated the elevated levels of albumin,NHCl₄-induced urea, and CCl₄-induced GOT, GPT, and ALP in the mediumafter induction (FIG. 14A). LDL uptake assay revealed that these cellscontained ability of LDL uptake (FIG. 14B). Oil-O-Red staining revealedthe presence of lipid droplets, suggesting the capacity in adipogenesis(FIG. 14C). Furthermore, glycogen storage test revealed a positiveperiodic acid-Schiff (PAS) staining, which was supported by diastasedigestion and PAS fluorescence emission test in either cytology orhistology (FIG. 14D). Next, qPCR analysis revealed the capacity ofcytochromes P450 (CYPs) enzymes in response to a variety of metabolizespecific drugs, including CYP3A4, CYP7A1, CYP2B6, CYP1A2, CyP2C8,CYP2C9, CYP2D6, and CYP2E1 (FIG. 14E). Functionally, for example, therefampin-induced CYP3A4 mRNA was reduced by its inhibitor itraconazole;while the 2,4,6-trihydroxyacetophenone (THA)-induced cholesterol7α-hydroxylase (CYP7A1) mRNA was reduced by chenodeoxycholic acid(CDCA). These results suggest the capability in oxidation of xenobioticsas well as the bile acid and cholesterol metabolism, respectively.

8) Functional Hepatocyte-Like Cells Posses Characteristic of Stem CellHoming

Animal study was designed to mimic the clinical scenario in acutehepatic failure, by which intraperitoneal infusion of carbontetrachloride (CCl₄) was given in Sprague Dawley rats, followed byintravenous injection (tail vein) of the hTS cell-derivedhepatocyte-like cells. The goals were to examine whether thesexenografts can be survival by homing in the rat's liver and what role ofthese cells play. Serum samples were collected at 0, 1, 2, 4, and 7 daysto measure AST and ALT levels. Rats were sacrificed at 2, 4, and 7 daysto get the liver samples for histopathological studies. Biochemicalstudy revealed that serum AST and ALT levels appeared to besignificantly higher in the cell therapy group (CCl₄+cells) than thecontrol group (CCl₄ only) over time (FIG. 15A). To elucidate thisambiguous observation, the liver tissues were inspectedhistopathologically.

Using human cytoplasmic marker stem-121™ (Stem Cell Technologies. Inc.WA) as an indicator, a positive immunoreactive stem-121 expression inhTS cells was identified (FIG. 15B), followed by the confirmation ofpresence of stem-121-positive hepatocyte-like cells in the liver tissues4-day after implantation (FIG. 15C). This observation indicated thatintravenous administration of the hepatocyte-like cells enabled to behoming to the liver tissues. Interestingly, these stem-121^(positive)hepatocytes underwent degeneration as well immunohistochemically (FIG.15C). This fact explains a much higher elevation of AST and ALT levelsin the cell therapy group than the only CCl4-treated group because of anadditional effect of CCl₄ (24 hr half-live in blood) which causeddegeneration of the implanted hepatocyte-like cells. The intravenousinfusion of hTS cell-derived hepatocyte-like cells can reach to andreside in the CCl₄ injury liver tissues.

9) Hepatocyte-Like Cells Possess Immune Privilege by Expressing HLA-Gand TGFβ1 and Recruiting Capacity of CD4⁺Foxp3⁺ Treg Cells

Implanted hepatocyte-like cells expressed human leukocyte antigen G(HLA-G) because there was a coexpression of stem-121 and HLA-G in thehepatic tissues at 4-days post-implantation immunohistochemically (FIG.15D), suggesting a characteristic of immune privilege. HLA-G,membrane-bound or soluble, strongly acts on different immune cell types(NK, T, B, monocytes/dendritic cells) to inhibit both innate andadaptive immunity through the interaction with inhibitory receptors thatare expressed at the surface of immune cells.

Furthermore, hepatocyte-like cells were able to secrete TGFβ1 into theECMs (FIG. 13B). TGFβ1 is a critical regulator of thymic T celldevelopment and a crucial player in peripheral T cell homeostasis,tolerance to self antigens, and T cell differentiation during the immuneresponse. Meanwhile, CD4⁺Foxp3⁺ T regulatory (Treg) cells were presentin the hepatic sinusoids immunocytochemically (FIG. 15E), suggesting therecruitment of the Treg cells to the liver in response to the implantedcells. Active immune suppression by cytokine TGFβ1 or CD4⁺Foxp3⁺ Tregcells plays a pivotal mechanism of peripheral T cell tolerance. Theimplanted hepatocyte-like cells possess immune privilege by expressingHLA-G and TGFβ1, and recruiting CD4⁺Foxp3⁺ Treg cells to the liver tocontrol peripheral T cell tolerance.

TABLE 1 Antibodies used, e.g., in Example 1. Target Manufacturer Codeno. WB Flow IF FGFR1 Abcam ab10646 1/400 1/200 MIXL1 Abcam ab578541/1000 1/200 SMAD4 Santa Cruz sc-7966 1/200 Biotechnology CDX2 AbcamAb76541 1/1000 1/40 1/200 Cell signaling 3977s 1/1000 BD Pharmingen560395 1/40 OCT4 Abcam Ab19857 5 μg/ml 1 μg/ml Millpore MAB4419 2 μg/ml1/200 BD Pharmingen 560794 (Cy 5.5) 1/40 NANOG Chemicon AB9220 1/10001/200 Cell signaling 3580s 1/1000 1/200 BD Pharmingen 560791 1/40 SOX2Epitomics 2683s 1/1000 Abcam Ab59776 1/1000 1/200 BD Pharmingen 560291(PE) 1/40 C-peptide Abcam Ab14181 1/100 1/50 Santa Cruz Sc-51647 1/1001/50 Biotechnology β-actin Santa Cruz Sc-130065 1/2000 BiotechnologySomatostatin Santa Cruz Sc-55565 1/200 1/100 Biotechnology Glut2Chemicon AB1342 1/100 Glucagon Santa Cruz Sc-13091 1/200 1/100Biotechnology Insulin Santa Cruz sc-7839 1/100 1/50 Biotechnology Ngn3Santa Cruz sc-25654 1/200 1/100 Biotechnology Amylase Santa Cruzsc-12821 1/200 1/100 Biotechnology Pancreatic Chemicon sc-80494 1/100polypeptide PDX1 BD Pharmingen 562160 1/2000 1/200 Cell signaling 56791/1000 1/200 PI3K Cell signaling 4249s 1/1000 p-AKT(Ser473) Cellsignaling 4058 1/1000 AKT Cell signaling 4685 1/1000 CREB1 Cellsignaling 9197 1/1000 p-CREB1(Ser1330) Cell signaling 9191 1/1000β-Catenin Cell signaling 9587 1/1000 Epitomics 1247-1 1/2000 GSK3β Cellsignaling 9315 1/1000 p-GSK3β(Ser9) Cell signaling 9336 1/500 GSK-3α/β(Tyr- ECM Biosciences GM1321 1/500 279/Tyr-216) GSC Abcam ab117871 1/5001/100 Brachyury Abcam Ab20680 1/1000 1/200 SOX17 Origene TA502483 1/2001/100 FOXA2 Abcam Ab40874 HNF1b Abcam ab59118 1/1000 1/200 SOX9 Abcamab26414 1 μg/ml 1/200 PTF1a Abcam ab57257 1/500 1/200 NKX6.1 Abcamab90716 1 μg/ml 5 μg/ml GATA4 Abcam ab84593 1/1000 1/200 NKX2.2 AbcamAb79916 1/1000 1/200 FOXA2 Abcam Ab40874 1/1000 1/200 SOX17 R&D systemsAF1924 1/1000 1/200 CXCR4 Abcam Ab124824 1/1000 1/200 HNF4a Santa CruzSc374229 1/200 1/100 1/100 Biotechnology Albumin Calbiochem 126584 1/5001/100 1/100 AFP Millipore Mabd78 1/1000 1/100 1/200 BSEP Santa CruzSc74500 1/200 1/100 1/100 Biotechnology MRP2 Santa Cruz Sc5570 1/2001/100 1/100 Biotechnology Betatrophin Acris Antibodies GmbH H00055908-1/500 1/100 1/100 B01P

TABLE 2 Primers used, e.g., in Example 1 SEQ SEQ NCBI Target ID IDaccession gene Gene Name Forward NO: Reverse NO: numbers T Brachyuryacctggg 9 actgact 58 NM_001270484.1 tactccc ggagctg aatccta gtaggt SOX7Sex cgaagcg 10 ccacgac 59 NM_031439.3 determining aggcgac tttcccaregion Y cc gcatct (SRY)-box 7 CXCR4 C-X-C gaaaccc 11 agtagtg 60NM_001008540.1 chemokine tcagcgt ggctaag receptor ctcagt ggcaca type 4FOXA2 Forkhead ctggtcg 12 ggaggag 61 NM_021784.4 box protein tttgttgtagccct A2 tggctg cgg SOX17 SRY-box 17 gatacgc 13 acgactt 62 NM_022454.3cagtgac gcccagc gaccag atcttg AFP Alpha-1- cagccac 14 ggccaac 63NM_001134.2 fetoprotein ttgttgc accaggg caactc tttact ALB Albuminaagcctt 15 gcacagc 64 NM_000477.5 ggtgttg agtcagc attgcc catttc HNF1aHepatocyte caccaag 16 tctcgat 65 NM_000545.5 nuclear caggtct gacgctgfactor tcacctc tggttg 1-alpha HNF4a Hepatocyte tgacgat 17 agcccgg 66NM_178850.2 nuclear gggcaat aagcatt factor 4- gacacg tcttga alpha HNF6Hepatocyte gcttagc 18 ctgacag 67 NM_004498.2 nuclear agcatgc tgctcagfactor 6 aaaagga ctccaa TTR transthyretin gcctctg 19 atcccat 68NM_000371.3 ggaaaac ccctcgt cagtga ccttca KRT8 Keratin 8 ggacctg 20tctggtt 69 NM_001256282.1 caggaag gaccgta ggatct actgcg KRT18 Keratin 18acatccg 21 tccaagc 70 NM_199187.1 ggcccaa tggcctt tatgac cagattt KRT19Keratin 19 agctgag 22 gatcttc 71 NM_002276.4 catgaaa ctgtccc gctgccttcgagca SERPINA1 Serpin tccgata 23 agacggc 72 NM_000295.4 peptidaseactgggg attgtcg inhibitor, tgacct attcact clade A (alpha-1antiproteinase, antitrypsin), member 1 TAT Tyrosine gatgagc 24 acagtag73 NM_000353.2 aminotransferase agcaaag ggtcccc gcaacc aatgga G6PCGlucose-6- tcaacct 25 gtataca 74 NM_000151.3 phosphatase cgtcttt cctgctgaagtgga tgcccat tt ADH1C Alcohol gctgcag 26 ccccgag 75 NM_000669.4dehydrogenase gaatctg gattgcc 1C (class tcgttc tagatca I), gamma tpolypeptide APOF Apolipoprotein aatgact 27 caggaca 76 NM_001638.2 Fggactgt aggggtc gtgggta tgagga C/EBPa CCAAT- taactcc 28 atgtcga 77NM_004364.4 enhancer- cccatgg tggacgt binding agtcgg ctcgtgprotein alpha C/EBPb CCAAT- actttag 29 gatttaa 78 NM_005194.3 enhancer-cgagtca aggcagg binding gagccg cggcg protein beta CPS1 Carbamoyl-aggccca 30 agcaaca 79 NM_001122633.2 phosphate tgccaca gaggatgsynthase 1, aatca gatggc mitochondrial PCK2 Phosphoenol acagtga 31ccgcaca 80 NM_001018073.2 pyruvate aggtcga taccagg carboxykinase 2,ctccg tttcca mitochondrial TDO2 Tryptophan tgggaac 32 tcggtgc 81NM_005651.3 2,3- tacctgc atccgag dioxygenase atttgga aaacaa GYS2Glycogen ccaagag 33 tgcctcc 82 NM_021957.3 synthase 2 aagctac aacttta(liver) caaagcc ttggtca c HHEX Hexosaminidase cccctgg 34 tctcctc 83NM_002729.4 A (alpha gcaaacc catttag polypeptide) tctact cgcgtc PROX1Prospero agcaaat 35 ctcttgt 84 NM_001270616.1 homeobox 1 gactttg aggcagtaggttcc tcgggg a CX32 Connexin 32 gctcccc 36 actagga 85 NM_000166.5aaggtgt tgagctg gaatga caggga BSEP Bile salt tattcac 37 agaagcc 86NM_003742.2 export pump agggtcg aactcta ttggct acgcca MRP2 Multidruggtgtttc 38 ccaggtt 87 NM_000392.4 resistance- cacagag cacatct associatedcggcta cggact protein 2 CYP1A2 Cytochrome aacaagg 39 ggaagag 88NM_000761.4 P450 1A2 gacacaa aaacaag cgctgaa ggctgag t t CYP2B6Cytochrome atggggc 40 agaggcg 89 NM_000767.4 P450 2B6 actgaaa gggacacaagactg tgaatga a c CYP3A4 Cytochrome ccttaca 41 agctcaa 90 NM_017460.5P450 3A4 catacac tgcatgt acccttt acagaat ggaagt ccccggt ta CYP2C8Cytochrome tatggtc 42 tcaactc 91 NM_001198855.1 P450 2C8 ctgtgtt ctccacacaccgt aggcagt CYP2C9 Cytochrome ttcatgc 43 ttgcaca 92 NM_000769.2P450 2C9 ctttctc gtgaaac agcagg atagga CYP2C19 Cytochrome cgaggtc 44tgtcatg 93 NM_000771.3 P450 2C19 cagagat tagcaca acatc gaagtg CYP2D6Cytochrome ctaaggg 45 ctcacca 94 NM_000106.5 P450 2D6 aacgaca ggaaagcctcatca aaagaca c c CYP2E1 Cytochrome acagaga 46 atgagcg 95 NM_000773.3P450 2E1 ccaccag gggaatg cacaact acacaga CYP3A5 Cytochrome gaagaaa 47aagaagt 96 NM_000777.4 P450 3A5 agtcgcc ccttgcg tcaac tgtcta CYP7A1Cytochrome tgctact 48 tccgtga 97 NM_000780.3 P450 7A1 tctgcga gggaattaggcat caaggc UGT UDP cccctat 49 attgatc 98 NM_019077.2 glucuronosyltttttca ccaaaga transferase aaaatgt gaaaacc ctt ac BetatrophinChromosome acatctc 50 tgctctg 99 NM_018687.6 19 Open cctcccc tgctcagagactc aagtgg Reading ctgtcgg 51 gagtctg 100 Frame 80 ctgaggg gggaggg(Angiopoietin- tttccat agatgt Like Protein 8, Hepatocellular Carcinoma-Associated Gene TD26, Lipasin,) miR12 tctgcgg 52 tctgcct 101NC_000008.11 4-2 ctctttg tcagcac ChIP gtttca aagagg gcggctc 53 ctgcctt102 tttggtt cagcaca tcaagg agagga miR12 cccgcag 54 agaaggg 103NC_000020.11 4-3 ttctcaa agccagg ChIP ggacac caagtc SOX17 ttgtaga 55gtgaagc 104 NC_000008.10 ChIP ttgctct cttggct ctctcct agggg cc FOXA2cccatca 56 ttgggag 105 NC_000020.10 ChIP ttgattc gctgaga ctggat tttgtcBetatrophin gtcagcc 57 catgtgg 106 NC_000019.9 ChIP ctccctg atttccaactgat gcctgc

TABLE 3 Transcriptional gene profiles throughout hepatic differentiationHepatic Hepatocy- endoderm Hepatoblast Hepatoblast like cellsFunction\Stage Gene DE (8 hr) (8 hr to 1 D) 2 D 4 D (>4 D) DE CXCR4+++ + + + NA Foxa2 ++++ + ++ + Sox17 ++++ ++ + + HHEX ++ + − +++Brachyury (T) ++++ + − − Sox7 ++++ ++ − + NA Tyrosine catabolism TAT NA++ − − − Fetal α-fetoprotein AFP NA + − ++++ +++ precursor Proteinscarrier ALB NA + + ++++ ++++ synthesized in the liver GluconeogenesisPCK2 NA + + ++ +++ Pancreatic β-cell Betatrophin NA + + ++ +++ promoter,Lipid regulator Serine protease SERPINA1 NA + − ++ +++ inhibitor Bileacid biosynthesis CYP7A1 NA + + + +++ Drug and steroid CYP2B6 NA + − +++++ metabolism (phase I) Drug and steroid CYP3A4 NA + + ++ +++metabolism (phase I) Ethanol catabolism ADH1C NA + + ++ ++ (phase I)Liver glucagon GYS2 NA + + ++ ++ synthase PEPCK NA + − ++ − Hepatictranscriptional Hnf6 NA + + + ++ activator Secretion of bile salts BSEPNA + − + ++ Cholesterol transport APOF NA + − − ++ regulator Hepatic gapjunction Cx32 (GJB1) NA + − + ++ Regulator of several Hnf4α NA + − + +hepatic genes Adipocyte C/EBPβ NA + + − + differentiation Thyroxin- andretinol- TTR NA + − − + binding protein Enzyme of urea cycle CPS1 NA +− + + Enzyme of glucose G6PC NA − − +++ +++ homeostasis Regulator ofseveral Hnf1α NA − − ++ +++ hepatic genes IL-6-mediated barrier CK18 NA− − ++ ++ protection IL-6-mediated barrier CK8 NA − − ++ ++ protectionHepatocyte migration PROX1 NA + − + +++ Organization of bile CK19 NA −− + ++ duct Hepatobiliary excretion MRP2/ NA + − − + ABCC2 Tryptophanmetabolism TDO2 NA + ++ − + Denotation: NA, not available; (−)indicating expression <2-fold, (+) >2-fold, (++) >10-fold,(+++) >100-fold, and (++++) >1,000-fold.

TABLE 4 Antibodies used, e.g., in Example 2 Target Manufacturer Code no.WB Flow IF/IHC Stem121 StemCells AB-121-U-050 1/1000 Mixl1 Abcam ab578541/1000 1/200 Smad4 Santa Cruz sc-7966 1/200 Biotechnology Cdx2 AbcamAb76541 1/1000 1/40 1/200 Cell signaling 3977s 1/1000 BD Pharmingen560395 1/40 Oct4 Abcam Ab19857 5 μg/ml 1 μg/ml Millipore MAB4419 2 μg/ml1/200 BD Pharmingen 560794 (Cy 1/40 5.5) c-Kit Santa Cruz Sc19983 1/400Biotechnology CK18 Abcam Ab32118 1/100 1/200 CK19 Cell signaling 45581/200 β-actin Santa Cruz Sc-130065 1/2000 Biotechnology ADH Santa CruzSc137078 1/200 Biotechnology CPS1 Abcam Ab110303 1/200 ASGR1 ThermoFisher MAB0244 1/100 1/200 Scientific Cx32 Santa Cruz Sc7258 1/100Biotechnology ApoF Santa Cruz Sc107409 1/400 Biotechnology CYP1A1 SantaCruz Sc25304 1/100 Biotechnology CYP2B6 Santa Cruz Sc62204 1/100Biotechnology HLA-G Abcam Ab4570 1/200 CD4 eBioscience 14-0040-85 1/400Foxp3 Abcam Ab22510 1/400 HNF4A Santa Cruz Sc374229 1/200 1/100Biotechnology α-tubulin GeneTex GTX112141 1/1000 CREB1 Cell signaling9197 1/1000 p- Cell signaling 9191 1/1000 CREB1(Ser1330) GSK3β Cellsignaling 9315 1/1000 TGFβ1 Santa Cruz Sc146 1/500 Biotechnology COL4Santa Cruz Sc59814 1/500 Biotechnology FN1 Santa Cruz Sc6952 1/500Biotechnology GSC Abcam ab117871 1/500 1/100 Brachyury Abcam Ab206801/1000 1/200 Sox17 Origene TA502483 1/1000 1/100 R&D MAB1927 Sox7 SantaCruz Sc20093 1/200 Biotechnology Foxa2 Abcam Ab40874 1/1000 1/200 GATA4Abcam ab84593 1/1000 1/200 CXCR4 Abcam Ab124824 1/1000 1/200 Hnf4a SantaCruz Sc374229 1/200 1/100 1/100 Biotechnology Albumin Calbiochem 1265841/500 1/100 1/100 AFP Millipore Mabd78 1/1000 1/100 1/200 BSEP SantaCruz Sc74500 1/200 1/100 1/100 Biotechnology MRP2 Santa Cruz Sc55701/200 1/100 1/100 Biotechnology Betatrophin Acris Antibodies GmbHH00055908- 1/500 1/100 1/100 B01P

TABLE 5 Primers used, e.g., in Example 2 SEQ SEQ Target ID ID geneForward NO: Reverse NO: Brachyury acctgggtactcccaatccta  9actgactggagctggtaggt  58 Sox7 cgaagcgaggcgaccc 10 ccacgactttcccagcatct 59 Cxcr4 gaaaccctcagcgtctcagt 11 agtagtgggctaagggcaca  60 Foxa2ctggtcgtttgttgtggctg 12 ggaggagtagccctcgg  61 Sox17 gatacgccagtgacgaccag13 acgacttgcccagcatcttg  62 AFP cagccacttgttgccaactc 14ggccaacaccagggtttact  63 Albumin aagccttggtgttgattgcc 15gcacagcagtcagccatttc  64 Hnf1a caccaagcaggtcttcacctc 16tctcgatgacgctgtggttg  65 Hnf4a tgacgatgggcaatgacacg 17agcccggaagcatttcttga  66 Hnf6 gcttagcagcatgcaaaagga 18ctgacagtgctcagctccaa  67 Ttr gcctctgggaaaaccagtga 19atcccatccctcgtccttca  68 KRT8 ggacctgcaggaagggatct 20tctggttgaccgtaactgcg  69 KRT18 acatccgggcccaatatgac 21tccaagctggccttcagattt  70 KRT19 agctgagcatgaaagctgcct 22gatcttcctgtccctcgagca  71 Serpina1 tccgataactggggtgacct 23agacggcattgtcgattcact  72 Tat gatgagcagcaaaggcaacc 24acagtagggtccccaatgga  73 G6p tcaacctcgtctttaagtggatt 25gtatacacctgctgtgcccat  74 Adh1C gctgcaggaatctgtcgttc 26ccccgaggattgcctagatcat  75 ApoF aatgactggactgtgtgggta 27caggacaaggggtctgagga  76 C/EBPa taactcccccatggagtcgg 28atgtcgatggacgtctcgtg  77 C/EBPb actttagcgagtcagagccg 29gatttaaaggcaggcggcg  78 Cps1 aggcccatgccacaaatca 30 agcaacagaggatggatggc 79 Pck2 acagtgaaggtcgactccg 31 ccgcacataccaggtttcca  80 Tdo2tgggaactacctgcatttgga 32 tcggtgcatccgagaaacaa  81 Gys2ccaagagaagctaccaaagcc 33 tgcctccaactttattggtcac  82 Hexcccctgggcaaacctctact 34 tctcctccatttagcgcgtc  83 Prox1agcaaatgactttgaggttcca 35 ctcttgtaggcagttcgggg  84 Cx32gctccccaaggtgtgaatga 36 actaggatgagctgcaggga  85 BSEPtattcacagggtcgttggct 37 Agaagccaactctaacgcca  86 MRP2gtgtttccacagagcggcta 38 Ccaggttcacatctcggact  87 CYP1A2aacaagggacacaacgctgaat 39 ggaagagaaacaagggctgagt  88 CYP2B6atggggcactgaaaaagactga 40 agaggcggggacactgaatgac  89 CYP3A4ccttacacatacacaccctttggaagt 41 agctcaatgcatgtacagaatccccggtta  90 CYP2C8tatggtcctgtgttcaccgt 42 tcaactcctccacaaggcagt  91 CYP2C9ttcatgcctttctcagcagg 43 ttgcacagtgaaacatagga  92 CYP2C19cgaggtccagagatacatc 44 tgtcatgtagcacagaagtg  93 CYP2D6ctaagggaacgacactcatcac 45 ctcaccaggaaagcaaagacac  94 CYP2E1acagagaccaccagcacaact 46 atgagcggggaatgacacaga  95 CYP3A5gaagaaaagtcgcctcaac 47 aagaagtccttgcgtgtcta  96 CYP7A1tgctacttctgcgaaggcat 48 tccgtgagggaattcaaggc  97 UGTcccctattttttcaaaaatgtctt 49 attgatcccaaagagaaaaccac  98 Betatrophinacatctccctccccagactc 50 tgctctgtgctcagaagtgg  99 ctgtcggctgagggtttccat51 gagtctggggagggagatgt 100 miR124-2 tctgcggctattggtttca 52tctgccttcagcacaagagg 101 ChIP gcggctctttggtttcaagg 53ctgccttcagcacaagagga 102 miR124-3 cccgcagttctcaaggacac 54agaagggagccaggcaagtc 103 ChIP Sox17 ttgtagattgctctctctcctcc 55gtgaagccttggctagggg 104 ChIP Foxa2 cccatcattgattcctggat 56ttgggaggctgagatttgtc 105 ChIP betatrophin gtcagccctccctgactgat 57catgtggatttccagcctgc 106 -2.5 ChIP kb

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

What is claimed is: 1-89. (canceled)
 90. An isolated human hepatocyte,wherein the isolated human hepatocyte expresses human leukocyte antigenG (HLA-G) and human cytoplasmic marker stem 121 (stem 121).
 91. Theisolated human hepatocyte of claim 90, wherein the isolated humanhepatocyte is a human hepatic progenitor cell.
 92. The isolated humanhepatocyte of claim 90, wherein the isolated human hepatocyte furtherexpresses one or more biomarkers selected from the group consisting ofCXCR4, FOXA2, SOX17, HHEX, TTR, ALB, TAT, CYP7A1, BSEP, SERPINA1, G6PC,ABCC2, C/EBPβ, HNF1α, HNF4α, and any combination thereof.
 93. Theisolated human hepatocyte of claim 90, wherein the isolated humanhepatocyte further expresses TGFβ1, fibronectin, or collagen IV inextracellular matrix (ECM).
 94. The isolated human hepatocyte of claim90, wherein the isolated human hepatocyte recruits CD4⁺Foxp3⁺ Tregcells.
 95. The isolated human hepatocyte of claim 90, wherein theisolated human hepatocytes form tissue of a 3-dimensional structure. 96.The isolated human hepatocyte of claim 90, wherein the isolated humanhepatocytes cluster or aggregate.
 97. The isolated human hepatocyte ofclaim 90, wherein the isolated human hepatocytes form a crescent cellmass.
 98. The isolated human hepatocyte of claim 90, wherein theisolated human hepatocyte further expresses one or more markers selectedfrom the group consisting of TGFβ1, C-kit, CK19, CK18, ALB, α-AFP,betatrophin, ADH1, APOF, CPS1, GATA4, CYP1A1, CYP2B6, ASGR1, CXCR4,BSEP, MRP2, Cx32, and any combination thereof.
 99. A method of screeninga therapeutic compound for use in treatment or prevention of acondition, comprising: a) contacting the isolated human hepatocyte ofclaim 90, with the therapeutic compound; and b) detecting an expressionlevel of a biomarker in the isolated human hepatocyte.
 100. Acomposition comprising the isolated human hepatocyte of claim
 90. 101. Apharmaceutical composition comprising the isolated human hepatocyte ofclaim
 90. 102. A method of treating a condition in a subject, comprisingadministering to a subject a pharmaceutical composition that comprisesthe isolated human hepatocyte of claim 90, in an amount effective forthe isolated human hepatocytes to engraft to the subject.
 103. A methodof producing therapeutic proteins, comprising using a composition thatcomprises the isolated human hepatocyte of claim
 90. 104. A method ofregenerating a liver, comprising using a composition that comprises theisolated human hepatocyte of claim
 90. 105. A method of using genes in atherapy, comprising using a composition that comprises the isolatedhuman hepatocyte of claim
 90. 106. A method of bioprinting, comprisingusing the isolated human hepatocyte of claim
 90. 107. A method ofgenerating a tissue scaffold, comprising using the isolated humanhepatocyte of claim
 90. 108. An artificial tissue generated from theisolated human hepatocytes of claim
 90. 109. An artificial organgenerated from the isolated human hepatocytes of claim 90.